Methods and uses of encapsulated exudates and dried euglena biomass for binding metal

ABSTRACT

A method of binding a target metal in solution. The method of binding a target metal comprises contacting a solution containing i) a target metal with ii) an encapsulated exudate of a culture of algal flagellate, or a fraction thereof; or an encapsulated dried Euglena biomass or a fraction thereof, to form a complex between the target metal, and the encapsulated exudate or fraction thereof, or the encapsulated dried Euglena biomass or the fraction thereof; and optionally separating the complex from the solution. The disclosure also relates to a biosorbent element, as well as methods of using same in binding a metal in solution.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/482,952, filed Apr. 7, 2017, which is incorporated herein byreference in its entirety.

FIELD

The present disclosure relates to methods of binding metals, abiosorbent element, as well as methods of using the biosorbent elementin binding metals and water remediation. For example, the presentdisclosure relates to methods and uses of an encapsulated exudate of aculture of algal flagellate, or a fraction thereof, or an encapsulateddried Euglena biomass, or a fraction thereof, for binding metals inwater such as wastewater or mining process water.

BACKGROUND

Industrial wastewaters, generated from metal plating, mining, fertilizerproduction, tannery operations, battery production, pulp and paper, andpesticide production and application, are sources of heavy metal releaseinto the environment which not only pose an ecological threat, but alsomay present serious consequences for human health (Fu and Wang, 2011).Consequently, there exists a necessity for the efficient removal oftoxic metals from industrial effluent before any potential exposure tosurface and ground waters.

Mining operations have historically contributed to elevated levels ofmetals in the surrounding environment (Keller et al., 2007).Conventional methods of metal removal can include chemicalprecipitation, resin-based ion exchange, and activated carbons as wellas physical methods which utilize filtration, floatation, andcoagulation (Fu and Wang, 2011). These methods, however, can tend togenerate large capital and operational costs, substantial energyrequirements, and large volumes of toxic waste materials (Wang and Chen,2009). In addition, these procedures also tend to lack effectiveness atlower (<100 mg/L) concentrations and can become prohibitively expensivein terms of the volume of wastewater to be treated (Volesky, 2001).

As an alternative, the use of biological material for remediation ofindustrial wastewater, commonly referred to as biosorption and/orbioaccumulation, offers several advantages over conventional methodswhich include: ubiquity, the minimization of chemical and or biologicalsludge, operation over a broad range of physio-chemical conditions,relatively low capital investment and operational costs, and anincreased efficiency in the removal of contaminants from dilute effluent(Abbas et al., 2014; Malik, 2004).

Both living and non-living biomass have been successfully utilized formetal removal and have been studied extensively although the use ofnon-living biomass is becoming the preferred option in the majority ofrecent metal removal studies (Fomina and Gadd, 2014; Doshi et al., 2007;Kizilkaya et al., 2012; Kumar et al., 2015; Michalak et al., 2013). Theuse of non-living biotic material confers several benefits compared toutilizing metabolically active organisms such as eliminating nutritionalrequirements, toxicity thresholds, allowing for desorption and metalrecovery processes, and potentially improving sorption capacity throughan increase in the cell surface to area ratio (Donmez et al., 1999;Kadukova and Vircikova, 2005). Live algal cells are also limited interms of metal recovery as ions are bound intracellularly and/ormetabolic exudates may form complexes with metal ions outside the cellwhich serve to retain metals in the aqueous phase.

Non-living biological material has the potential to effectively removeas much as or greater amounts of metal ions from solution when comparedto living algal cells (Burdin and Bird, 1994; Tien et al., 2005). Thecharacteristics of non-living algae mentioned above contribute in largepart to the commercial viability of using such materials in remediationapplications in potentially remote settings where infrastructuredevelopment is constrained by physical and economic limitations.Additionally, using inert algal material as opposed to living stock formetal removal can result in altered surface chemistry that influencescation sorption (Tien et al., 2005). Cell surfaces of algal organismsconsist of an assortment of polysaccharides, proteins, and lipids all ofwhich include functional groups (e.g. carboxyl, amino, thiol, sulphateand hydroxyl groups) that are capable of binding metal ions (Crist etal., 1981). Modifications of the cell surface/structure can occur whenutilizing inactivation methods such as heat-drying, chemical treatments,and vacuum-drying which may result in increased or decreased sorptioncapacity and in this sense are dependent on the origin and pre-treatmentof the biological material (Gardea-Torresdey et al., 1990; Winter etal., 1994).

Euglena gracilis is a free-floating, flagellated unicellular species ofprotist which has been found to tolerate and accumulate heavy metals(Rodriguez-Zavala et al., 2007). It is a candidate for use inbioremediation of wastewaters (Mendoza-Cozatl et al., 2006; Olaveson andNalewajko, 2000).

SUMMARY

Accordingly, the present disclosure includes a method, comprising:

A method of binding a target metal, comprising:

-   -   contacting a solution containing    -   i) a target metal with    -   ii) an encapsulated exudate of a culture of algal flagellate, or        a fraction thereof; or    -   an encapsulated dried Euglena biomass or a fraction thereof,    -   to form a complex between the target metal, and the encapsulated        exudate or fraction thereof, or the encapsulated dried Euglena        biomass or the fraction thereof; and    -   optionally separating the complex from the solution.

The present disclosure also includes a biosorbent element comprising asubstrate carrying dried Euglena biomass, or a fraction thereof, or wetEuglena biomass, or a fraction thereof, or exudates of a culture ofEuglena, or a fraction thereof, in sufficient quantity to adsorb metalsfrom water passing therethrough, and methods of uses of the biosorbentelement in binding metals in water.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the disclosure, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will now be described in greater detail withreference to the drawings, in which:

FIG. 1 shows Fourier Transform Infrared Spectroscopy (FTIR) spectra ofdried Euglena biomass.

FIG. 2 shows Cu²⁺ sorption kinetics on non-living E. gracilis at initialconcentrations of (A) 20 ug·L⁻¹ and (B) 50 ug·L⁻¹ (C) 1 mg·L⁻¹ (D) 25mg·L⁻¹. (A) and (C) show pseudo-first-order (PFO) kinetic model. (B) and(D) show pseudo-second-order (PSO) kinetic model.

FIG. 3 shows Ni²⁺ sorption kinetics on non-living E. gracilis at initialconcentrations of (A) 1 mg·L⁻¹ and (B) 2 mg·L⁻¹ (C) 4 mg·L⁻¹ (D) 20mg·L⁻¹. (A) and (C) show pseudo-first-order (PFO) kinetic model. (B) and(D) show pseudo-second-order (PSO) kinetic model.

FIG. 4 shows Cu²⁺ sorption equilibrium on non-living E. gracilis. (A)shows Freundlich model and (B) shows Langmuir model.

FIG. 5 shows Ni²⁺ sorption equilibrium on non-living E. gracilis. (A)shows Freundlich model and (B) shows Langmuir model.

FIG. 6 shows Cu²⁺ and Ni²⁺ sorption equilibrium from binary-metalsolution on living E. gracilis. Error bars represent SE. Cu²⁺ in closedcircle and Ni²⁺ in open circle.

FIG. 7 shows sample sites.

FIG. 8 shows (A) a single packed column, and (B) packed columns inseries, with alginate beads inside.

FIG. 9 shows removal of target metals by bead WBS at (A) site A, and (B)site K.

FIG. 10 shows improved adsorption of Au after pretreatment.

FIG. 11 shows comparison of metal loading in (A) Series 1, and (B)Series 3, at site A.

FIG. 12 shows FTIR spectra for E. gracilis growing in (A) dark and (B)light conditions, with and without glucose supplementation inexponential and stationary phases. Trace (i): Euglena gracilis Medium(EGM) in exponential phase. Trace (ii): EGM+glucose in exponentialphase. Trace (iii): EGM in stationary phase. Trace (iv) EGM+glucose atthe stationary phase.

FIG. 13 shows molecular species seen at different stages of growth whenE. gracilis grows in (A) light and (B) dark conditions.

FIG. 14 shows the abundances of compound classes of E. gracilis grown indifferent culture conditions.

FIG. 15 shows the multivariate variation among cellular composition interms of elemental ratios. Vectors indicate the direction and strengthof each variable to the overall distribution.

FIG. 16 shows the multivariate variation among cellular composition interms of compound class abundances associated with E. gracilis grown inlight (open) and dark (black) conditions at (▪) exponential and (●)stationary phases. Vectors indicate the direction and strength of eachvariable to the overall distribution.

FIG. 17 shows representative fractograms for (A) light and (B) darkgrown cells. Replicates 1 and 2 are denoted by the red and blue linesrespectively. Blank runs are denoted by the black lines.

FIG. 18 shows the Venn diagrams of combined fractions and unfractionatedsupernatant grown in (A) light and (B) dark.

FIG. 19 shows the abundances of compound classes from cells grown inlight and dark conditions.

FIG. 20 shows the m/z distributions of light and dark combined fractionsand unfractionated portions of E. gracilis.

FIG. 21 shows the Venn diagrams for the m/z values in (A) light and (B)dark grown fractions.

FIG. 22 shows the abundances of compound classes found in AF4 fractionsisolated from cells grown in (A) light and (B) dark conditions.

FIG. 23 shows the multivariate variation among cellular composition ofAF4 fractions in terms of compound class abundances in light (open) anddark (closed) conditions. The combined and unfractionated samples areindicated by a triangle up and a triangle down, respectively. Vectorsindicate the direction and strength of each variable to the overalldistribution.

FIG. 24 shows bioluminescence response as a function of differentunfractionated algae exposed to 250 pM of Hg(NO₃)₂.

FIG. 25 shows the incorporation of non-living Euglena cells and/orexudates released by Euglena as binding sorbent for the passiveconcentration and removal of metals in a diffusive gradient in thinfilms (DGT) assembly.

FIG. 26 shows the performance of dialysis bag to retain small (SRFA),medium (Euglena exudates) and large compounds (Euglena cells).

FIG. 27 shows the Euglena exudates dialysate was slightly more enrichedcompared to SRFA.

FIG. 28 shows that no Euglena cells were noticeable in the dialysateafter 4 days (C). All original cells (A) were kept in the dialysis bagafter 4 days (B).

FIG. 29 shows change in metal sorption of the younger generation cellsgrown in normal (Nm) versus Hg-adapted Euglena gracilis cells.

FIG. 30 shows change in metal sorption of the older generation grown innormal (Nm) versus Hg-adapted Euglena gracilis cells.

FIG. 31 shows metal sorption ratio of the younger and older generationsof Hg-adapted Euglena gracilis cells.

FIG. 32 shows the FTIR spectra of Chelex (solid) and Euglena cells(dashed).

FIG. 33 shows the accumulated metal mass in unexposed DGT-Chelex andDGT-Euglena resin.

FIG. 34 shows the diffusion coefficient of DGT-Euglena binding resin at20° C.

DETAILED DESCRIPTION I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present disclosure herein described for which theyare suitable as would be understood by a person skilled in the art.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. The term “consisting” and its derivatives, as used herein,are intended to be closed terms that specify the presence of the statedfeatures, elements, components, groups, integers, and/or steps, butexclude the presence of other unstated features, elements, components,groups, integers and/or steps. The term “consisting essentially of”, asused herein, is intended to specify the presence of the stated features,elements, components, groups, integers, and/or steps as well as thosethat do not materially affect the basic and novel characteristic(s) offeatures, elements, components, groups, integers, and/or steps.

As used in this disclosure, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.In embodiments comprising an “additional” or “second” component, thesecond component as used herein is different from the other componentsor first component. A “third” component is different from the other,first, and second components, and further enumerated or “additional”components are similarly different.

As used in this disclosure, the term “algae” and its derivatives, asused herein, include photosynthetic microorganisms that are prokaryotesor eukaryotes. Photosynthetic microorganisms include photosyntheticmicroorganisms that are also capable of mixotrophic or heterotrophicgrowth.

As used in this disclosure, the term “algal flagellate” includes analgal microorganism with one or more flagella.

II. System, Methods and Uses

Dried algal flagellate biomass, or a fraction thereof, or an exudate ofa culture of algal flagellate, or a fraction thereof, can bind to metalsin water. Encapsulation of the dried algal flagellate biomass, or anexudate of a culture of algal flagellate provides a greater and moreconcentrated surface area for metal binding and also provides a methodfor the removal of dried algal flagellate biomass or an exudate of aculture of algal flagellate and the bound metals after the incubationperiod. The present examples demonstrated the usefulness ofspherificated dried algal flagellate biomass, wet algal flagellatebiomass, or a fraction thereof, or an exudate of a culture of algalflagellate, as well as a biosorbent element containing a substratecarrying a dried Euglena biomass, a wet Euglena biomass, or exudates ofEuglena, or a fraction thereof, in binding to metals.

Accordingly, the present disclosure includes a method, comprising:

A method of binding a target metal, comprising:

-   -   contacting a solution containing    -   i) a target metal with    -   ii) an encapsulated exudate of a culture of algal flagellate, or        a fraction thereof; or    -   an encapsulated dried Euglena biomass or a fraction thereof,    -   to form a complex between the target metal, and the encapsulated        exudate or fraction thereof, or the encapsulated dried Euglena        biomass or the fraction thereof; and    -   optionally separating the complex from the solution.

The term “solution” as used herein includes a homogeneous mixturecomposed of two or more substances.

The term “water” as used herein includes water in the form of asolution, suspension or slurry.

The term “non-living” as used herein refers to a microorganism beingdead, lifeless, or metabolically inactive, a condition inducedoptionally by radiation, including electromagnetic radiation andparticle radiation, biological treatment, chemical treatment, physicalforce, high hydrostatic pressure, or deprivation of necessities of life.

The term “exudate” as used herein refers to the secretion and/orexcretion from a microorganism into a media or any surrounding liquidwhere it lives or once lived. An exudate may be obtained by subjecting aculture of microorganism to solid liquid separation usingcentrifugation, wherein the exudate is recovered from the liquid mediaphase.

The fraction of algal flagellate biomass or an exudate of a culture ofalgal flagellate comprises any suitable fraction of algal flagellatebiomass or an exudate of a culture of algal flagellate for the bindingof metals.

The term “metal” as used herein includes metal ions, anionic metals,cationic metals, metalloids, alloys, rare earth elements, light metals,heavy metals, transition metals, base metals, ferrous metals, noblemetals and precious metals.

The metal can be any suitable metal which forms a complex with a driedalgal flagellate biomass, or a fraction thereof, a wet algal flagellatebiomass, or a fraction thereof, or an exudate of a culture of algalflagellate, or a fraction thereof. The expressions “form a complex”between the algal flagellate, an exudate of a culture of algalflagellate, or a fraction thereof and metal and “metal which forms acomplex” with the algal flagellate, an exudate of a culture of algalflagellate, or a fraction thereof, as used herein refers to forming acomplex between the metal and at least one compound that is a componentof the fraction of the algal flagellate or an exudate of a culture ofalgal flagellate. The metal can be an anionic or cationic metal such asarsenic, rare earth elements, and radionuclides. It will be appreciatedby a person skilled in the art that the non-living algal flagellate oran exudate of a culture of algal flagellate, or a fraction thereof,compounds with heteroatoms such as nitrogen (N), oxygen (O) and sulfur(S) that are capable of binding suitable metal ions. Non-living algalflagellate, an exudate of a culture of algal flagellate, or a fractionthereof is selected for binding a particular metal or class thereof. Forexample, Type-A metals typically form more stable complexes with O- andN-containing ligands whereas Type-B metals typically form more stablecomplexes with S-containing ligands and transition metals are known toexhibit behaviour intermediate between Type A and Type B metals. In someembodiments, the metal is a transition metal. For example, in someembodiments, the metal ion is a divalent transition metal (i.e. M²⁺) andoptionally is Hg²⁺ or a divalent transition metal that shows similarbinding to heteroatoms (e.g. N, O and S) such as Zn²⁺, Co²⁺, Ni²⁺ orPb²⁺. In another embodiment, the metal ion is Hg²⁺. In some embodiment,the metal is in a metal-cyano anionic complex.

The term “mining” as used herein includes an operation of extractingrare earth elements, aluminum, cobalt, copper, gold, molybdenum, nickel,palladium, platinum, rhodium, silver, uranium and/or zinc from theearth.

In one embodiment, the exudate may be obtained in a culture of anymicroorganism. In another embodiment, the exudate may be obtained in aculture of any algae.

In one embodiment, the metals are silver, gold, aluminum, arsenic,barium, beryllium, bismuth, calcium, cadmium, cobalt, chromium, copper,iron, potassium, lithium, magnesium, manganese, molybdenum, sodium,nickel, phosphorus, platinum, palladium, lead, antimony, selenium, tin,strontium, thallium, titanium, uranium, vanadium, tungsten, yttrium,zinc, scandium, lanthanum, rare earth element and divalent transitionmetals, optionally wherein the method binds a plurality of metalscomprising at least two or more of the foregoing.

The methods of binding metal ions in solution are useful for anysuitable use wherein it is desired to bind a metal ion in solution. Thesolution can be water, optionally wastewater, and the method is forremediation of water or wastewater having one or more metal to beremoved. For example, the method may be used for the remediation ofwastewater or mining process water as well as for other water treatmentand water purification applications. In an embodiment, the method is forremediation of wastewater or mining process water having a metal ion tobe removed and the water is wastewater or mining process water. Thewastewater can be any suitable wastewater. For example, the wastewatercan be domestic wastewater, urban wastewater, industrial wastewater orcombinations thereof. The mining process water can be any suitablemining process water. For example, the mining process water can be fromany mining operation involving the extraction of minerals or othergeological materials.

The term “industrial wastewater” includes any suitable water thatcontains metal ions and is waste from industry. For example, theindustrial wastewater can comprise metal processing effluent orwastewater from electroplating processes. For example, wastewaterstemming from the grinding of mineral and sediment can include dissolvedmetals such as divalent metals, for example, mercury which can be boundin the methods of the present disclosure. Accordingly, in an embodiment,the industrial wastewater comprises effluent from a mining operation.

The term “mining process water” or “mining solution” includes any waterused to leach metals of interest from rock or ore that contains thosemetals of interest in some concentration, or water at some point in amining process that contains metals of interest in some concentration.Mining process water or mining solution includes water from carbontrains, resin columns, ponds such as excess, intermediate, pregnant andtailing ponds, leach pads, electrowinning cells, tanks such asflotation, Merrill Crowe, solid phase extraction and pregnant tanks. Theskilled person readily recognizes sources of mining process water. In anembodiment, the methods described herein are for remediation or recoveryof metals from mining process water selected from carbon trains, resincolumns, ponds such as excess, intermediate, pregnant and tailing ponds,leach pads, electrowinning cells, tanks such as flotation, MerrillCrowe, solid phase extraction and pregnant tanks, using a biosorbentdescribed herein. In another embodiment, the methods described hereinare for remediation or recovery of metals from mining process waterentering or leaving carbon trains, resin columns, ponds such as excess,intermediate, pregnant and tailing ponds, leach pads, electrowinningcells, tanks such as flotation, Merrill Crowe, solid phase extractionand pregnant tanks, using a biosorbent described herein. In a specificembodiment, the methods described herein are for recovery of preciousmetals, optionally gold, silver, platinum and palladium, from miningprocess water selected from carbon trains, resin columns, ponds such asexcess, intermediate, pregnant and tailing ponds, leach pads,electrowinning cells, tanks such as flotation, Merrill Crowe, solidphase extraction and pregnant tanks, using a biosorbent describedherein. In another specific embodiment, the methods described herein arefor recovery of precious metals, optionally gold, silver, platinum andpalladium, from mining process water entering or leaving carbon trains,resin columns, ponds such as excess, intermediate, pregnant and tailingponds, leach pads, electrowinning cells, tanks such as flotation,Merrill Crowe, solid phase extraction and pregnant tanks, using abiosorbent described herein.

The methods of the present disclosure can also be used to capture ametal ion of interest from the water. For example, so that the metal ioncan be converted into the metal.

In an embodiment, the mining process water comprises a metal of interestat concentration of 0.1-2000 ppm, 1-1000 ppm, 5-800 ppm, or 10-600 ppm.In another embodiment, the mining process water comprises a metal ofinterest at concentration equal to or less than 2000, 1500, 1250, 1200,1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 ppm.

In an embodiment, the mining process water comprises a pH range of 2-13,or 8-11. In another embodiment, the mining process water comprises a pHof equal to or more than 2, 3, 4, 5, 6, 7, 8, 9 and 10. In anotherembodiment, the mining process water comprises a pH of equal to or lessthan 13, 12, 11, 10, 9, 8, 7 and 6.

In an embodiment, the mining process water was depleted of cyanide (CN).In another embodiment, the mining process water was treated withhydrogen peroxide. In another embodiment, the mining process water wastreated with hydrogen peroxide to deplete CN.

In an embodiment, the complex is separated from the solution. The methodof separation can involve any suitable means of separation and willdepend, for example, on the method by which the solution is contactedwith the encapsulated dried algal flagellate biomass, or a fractionthereof, or a dried algal flagellate biomass, or a fraction thereof, oran exudate of a culture of algal flagellate, or a fraction thereof. Inan embodiment, the separation comprises contacting the complex with amicroorganism or microorganism material to sequester the complex. Themicroorganism is any suitable microorganism that can uptake or sequesterthe complex. For example, gram-negative bacteria E. coli and any othersuitable microorganism are useful in this regard. The microorganismmaterial comprises any suitable microorganism material that can uptakeor sequester the complex. For example, materials from gram-negativebacteria E. coli and any other suitable microorganism are useful in thisregard.

In another embodiment, the algal flagellate is a Chlamydomonadaceae, aCryptophyceae, a Dinoflagellate, an Euglenaceae, a Haptophyta, ormixtures thereof. In another embodiment, the algal flagellate is aChlamydomonas sp., a Cryptophyta sp., a Dinophyta sp., an Euglena sp, ormixtures thereof. In another embodiment, the algal flagellate isChlamydomonas reinhardtii, Euglena gracilis, Euglena mutabilis, orcombinations thereof. In another embodiment, the algal flagellate isEuglena gracilis. In another embodiment, the algae flagellate is Euglenagracilis, Euglena mutabilis or combinations thereof. In a furtherembodiment, the algal flagellate comprises, consists essentially of orconsists of Euglena gracilis.

In another embodiment, the algae is a Chlorella sp., a diatom, acyanobacteria, a protist or mixtures thereof. In a further embodiment,the algae is Chlorella vulgaris, Chlamydomonas reinhardtii, Euglenagracilis, Euglena mutabilis, Scenedesmus obliquus, Thalassiosiraweissflogii or combinations thereof.

In another embodiment, the exudate of a culture of algal flagellatecomprises glutathione, metallothioneins, phytochelatins, polyphosphates,polysaccharides, or combinations thereof. It is an embodiment that theexudate of a culture of algal flagellate, or the encapsulated non-livingEuglena biomass, is spherificated or gelificated. In another embodiment,the spherification or gelification process comprises using anencapsulating immobilizing matrix to encapsulate the exudates of aculture of algal flagellate, or dried Euglena biomass in an immobilizingmatrix. In another embodiment, the immobilizing matrix comprises a resinor a polymer plastic. In another embodiment, the immobilizing matrixcomprises agar, agarose, alginate, carrageenan, cellulose, chitosan,polystyrene, polyurethane, polyvinyl, or combinations thereof. Inanother embodiment, the immobilizing matrix comprises sodium alginate.In another embodiment, the exudate of a culture of algal flagellate ishoused in a containment element. In another embodiment, the containmentelement comprises a semi-permeable membrane. In a further embodiment,the semi-permeable membrane comprises integral asymmetric membrane orthin film composite membrane.

The term “activated carbon” and its derivatives as used herein refers toa form of carbon processed to have small, low-volume pores, optionally0.3-3 cm³/g, that increase the surface area available for metaladsorption and/or chemical reactions. An activated carbon may beobtained from nutshells, coconut husk, peat, wood, coir, lignite, coal,and/or petroleum pitch.

In another embodiment, an exudate of a culture of algal flagellate isspherificated or gelificated and further mixed with activated carbon. Inanother embodiment, the activated carbon comprises nutshells, coconuthusk, peat, wood, coir, lignite, coal, and/or petroleum pitch.

As used in this disclosure, the term “encapsulate” and its derivatives,as used herein, refers to a state where a matter is surrounded by agelatinous substance(s), such as substantially surrounded or fullysurrounded. The process of encapsulation comprises spherification orgelification.

As used in this disclosure, the term “unencapsulated” and itsderivatives, as used herein, refers to a state where a matter is notencapsulated.

In an embodiment, an unencapsulated exudate of a culture of algalflagellate, or a fraction thereof, or an unencapsulated dried Euglenabiomass, or a fraction thereof, or an unencapsulated wet Euglenabiomass, or a fraction thereof, binds metals described herein in watersuch as wastewater or mining process water. In another embodiment, anunencapsulated exudate of a culture of algal flagellate, or a fractionthereof, or an unencapsulated dried Euglena biomass, or a fractionthereof, or an unencapsulated wet Euglena biomass, or a fractionthereof, is for use in remediation of wastewater having the metalsdescribed herein to be removed and the water is wastewater or miningprocess water, optionally before the wastewater or mining process watercontacts activated carbon.

As used in this disclosure, the terms “biosorbent” and “biosorbentelement” are used interchangeably.

In an embodiment, a biosorbent described herein comprises anencapsulated or unencapsulated microorganism, or an exudate of a cultureof microorganism described herein. In another embodiment, a biosorbentdescribed herein comprises an encapsulated or unencapsulatedmicroorganism, aquatic microorganism, algae, algal flagellate,Chlamydomonas sp., Cryptophyta sp., Dinophyta sp., Euglena sp,Chlamydomonas reinhardtii, Euglena mutabilis, Euglena gracilis,combinations thereof, a fraction thereof, or combinations of fractionsthereof, or an exudate of a culture of said microorganism, aquaticmicroorganism, algae, algal flagellate, Chlamydomonas sp., Cryptophytasp., Dinophyta sp., Euglena sp, Chlamydomonas reinhardtii, Euglenamutabilis, Euglena gracilis, combinations thereof, a fraction thereof,or combinations of fractions thereof.

In an embodiment, methods described herein comprise uses of anencapsulated or unencapsulated microorganism, or an exudate of a cultureof microorganism described herein. In another embodiment, methodsdescribed herein comprise an encapsulated or unencapsulatedmicroorganism, aquatic microorganism, algae, algal flagellate,Chlamydomonas sp., Cryptophyta sp., Dinophyta sp., Euglena sp,Chlamydomonas reinhardtii, Euglena mutabilis, Euglena gracilis,combinations thereof, a fraction thereof, or combinations of fractionsthereof, or an exudate of a culture of said microorganism, aquaticmicroorganism, algae, algal flagellate, Chlamydomonas sp., Cryptophytasp., Dinophyta sp., Euglena sp, or Chlamydomonas reinhardtii, Euglenamutabilis, Euglena gracilis, combinations thereof, a fraction thereof,or combinations of fractions thereof.

As used in this disclosure, the term “gelificate” and its derivatives,as used herein, is defined as the process of turning a substance into agelatinous form. This process comprises converting liquid substancesinto solids with the help of a gelling agent. Common gelling agents comefrom natural sources and include agar-agar, gelatin, carrageenan, gellangum, pectin and methylcellulose.

As used in this disclosure, the term “bead” and its derivatives, as usedherein, refers to encapsulated algal flagellate or exudates of a cultureof algal flagellate, or a fraction thereof.

In another embodiment, the pH of the beads is at least 2, 3, 4, 5, 6, or7. In another embodiment, the pH of the beads is at most 6, 7, 8, 9, 10,11, 12 or 13. In another embodiment, the pH of the beads is between 2and 13, optionally between 3 and 10, optionally between 4 and 7.

In another embodiment, the bead size is at least 0.1, 0.2, 0.3, 0.4,0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mm. In another embodiment, thebead size is at most 4, 5, 6, 7, 8, 9, or 10 mm. In another embodiment,the bead size is between 0.1 and 10, optionally between 2 and 6,optionally between 3 and 5.

As used in this disclosure, the term “dried biomass” and itsderivatives, as used herein, is defined as a biomass consists ofmoisture content of at most 25%.

In another embodiment, the moisture content of dried biomass is equal toor less than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11and 10 and 5%. In another embodiment, the moisture content of driedbiomass is between 5 and 25%, or between 10 and 20%. In anotherembodiment, the moisture content of dried biomass is below 10%, or below5%.

As used in this disclosure, the term “wet biomass” and its derivatives,as used herein, is defined as a biomass consists of moisture content ofmore than 25%.

In another embodiment, the moisture content of wet biomass is more than25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90%. In anotherembodiment, the moisture content of wet biomass is at least 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90%. In anotherembodiment, the moisture content of wet biomass is between 30 and 90%,between 50 and 85%, or between 70 and 80%.

In another embodiment, the amount or type of metal sequestered perweight of active material of the algal flagellate biomass is modified bypost-harvest treatment prior to spherification. In another embodiment,the post-harvest treatment comprises drying the biomass or fractionthereof. In another embodiment, the drying treatment comprises treatingthe biomass at a temperature of at least 45, 50, or 55° C. In anotherembodiment, the drying treatment comprises drying the biomass for atleast 24, 36, 48, 60, 72, 84 or 96 hours. In another embodiment, thedrying treatment comprises drying the biomass for about 72 hours at 50°C.

In another embodiment, the post-harvest treatment comprises heating thebiomass or fraction thereof. In another embodiment, the heat treatmentcomprises treating the biomass at a temperature of at least 56, 57, 58,59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 140,145, 150, 155 or 160° C. In another embodiment, the heat treatmentcomprises heating the biomass for at least 24, 36, 48, 60, 72, 84 or 96hours. In another embodiment, the heat treatment comprises heating thebiomass for about 72 hours at 80 or 120° C. In another embodiment, theheat treatment comprises heating the biomass for about 72 hours at 80 or120° C.

In another embodiment, the exudates of a culture of algal flagellate,the encapsulated dried Euglena biomass, the post-harvest treatedencapsulated dried Euglena biomass and/or the wet Euglena biomass,comprising different metal binding selectivities are housed in aplurality of columns, the columns arranged to produce a column effluent.In another embodiment, the plurality of columns is arranged in a seriescomprising a first column, a second column and a third column, and thesolution flows through the columns sequentially to produce the columneffluent. In another embodiment, the first column selectively binds tocopper, the second column selectively binds to silver, and the thirdcolumn selectively binds to gold. In another embodiment, the algalflagellate biomass or exudates of a culture of algal flagellatecomprises selectivity for lead, copper, silver and/or gold. In anotherembodiment, the algal flagellate biomass or exudates of a culture ofalgal flagellate comprises selectivity for a metal described herein.

In another embodiment, a pre-treatment of the solution is performed bymixing the solution with an appropriate amount of either thespherificated algal flagellate biomass or free algal flagellate,followed by the removal of the spherificated algal flagellate biomass orfree algal flagellate. In another embodiment, the pre-treated solutionis passed through a column or a series of columns containingspherificated algal flagellate biomass or an exudate of a culture ofalgal flagellate.

In an embodiment, the biosorbent described herein has the loadingcapacity of at least 0.01, 0.1, 1, 10, 100, 1000, 1×10⁴, 1×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, or 5×10⁶ gram metal described herein per tonnebiosorbent.

As used in this disclosure, a “desorbent” is a solution that is used torelease a substance (for example, a target metal) from the structurethat it is bound to (for example, an encapsulated biomass, exudates, ora fraction thereof).

Desorption is carried out to liberate/reactivate the binding sites ofthe structure, which allows for the collection of the target metal insolution and the ability for further binding (i.e. adsorption) by theencapsulated exudates/biomass to a target metal in a solution,preferably fresh solution. A successful desorption or desorbent canaccomplish the foregoing advantages, while limiting the effect on thestability and/or integrity of the adsorptive structure. Desorbents canoptionally include acids (e.g. nitric acid, hydrochloric acid, sulphuricacid, citric acid), bases (e.g. sodium hydroxide, sodium carbonate,sodium bicarbonate), chelating agents (e.g. EDTA), or other compounds(e.g. thiourea, potassium cyanide, sodium citrate, sodium nitrate).Biosorbent described herein can optionally undergo multiple rounds orcycles of desorption and adsorption while retaining stability and/orintegrity of the adsorptive structure. The skilled person readilyrecognizes alternate desorbents suitable for desorption of a targetmetal from a biosorbent described herein.

In an embodiment, the methods described herein further comprisedesorbing the target metal. In another embodiment, the desorbing of thetarget metal comprises contacting the complex with a desorbent. Inanother embodiment, the desorbent comprises an acidic or basic solution.In another embodiment, the desorbent comprises potassium cyanide orthiourea. In another embodiment, the acidic solution comprises one ormore of hydrochloric acid, nitric acid, sulphuric acid, and citric acid.In another embodiment, the basic solution comprises one or more ofsodium hydroxide, sodium carbonate, and sodium bicarbonate. In anotherembodiment, the target metal is a precious metal, optionally gold,silver, platinum or palladium. In an embodiment, a desorbent describedherein desorbs at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% of a target metal described hereinfrom a biosorbent described herein. In another embodiment, biosorbentsdescribed herein retain at least 70%, 75%, 80%, 85%, 90%, 91%%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% binding capacity to a targetmetal described herein, after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 rounds or cycles ofdesorption and adsorption. In another embodiment, the desorption methodsdescribed herein retain at least 70%, 75%, 80%, 85%, 90%, 91%%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% binding capacity to a targetmetal described herein, after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 rounds or cycles ofdesorption and adsorption.

As used herein, the term “fresh” refers to a new or different batch ofsolution and does not necessarily mean that the solution is freshlymade, acquired, obtained, or retrieved from a source. In anotherembodiment, after desorbing the target metal from the complex, theencapsulated exudate of a culture of algal flagellate or a fractionthereof, the encapsulated dried Euglena biomass or a fraction thereof,or the encapsulated wet Euglena biomass or a fraction thereof, contactsa fresh solution containing a target metal. In an embodiment, a freshsolution comprises mining process water or mining solution.

As used in this disclosure, the term “selective” and its derivatives, asused herein, refers to a preference towards one metal over others.

In an embodiment, the algae is grown in light in the absence or presenceof glucose supplement. In an embodiment, the algae is grown in the darkin in absence or presence of glucose supplementation. In anotherembodiment, the algae is grown in the light for 24, 48 or 72 h untilreaching exponential growth phase. In another embodiment, the algae isgrown in the dark for 24, 48 or 72 h or until reaching exponentialgrowth phase. In yet another embodiment, the glucose supplementationcomprises a range of 0.1 to 20 g·L⁻¹ glucose.

In an embodiment, a biosorbent element contains a substrate carrying adried Euglena biomass, or an exudate of a culture of Euglena, or afraction thereof, in sufficient quantity to adsorb metals from waterpassing therethrough. In another embodiment, the biosorbent element is abiosorbent diffusive gradient across a plurality of thin films. In afurther embodiment, the biosorbent element binds metals comprise silver,gold, aluminum, arsenic, barium, beryllium, bismuth, calcium, cadmium,cobalt, chromium, copper, iron, potassium, lithium, magnesium,manganese, molybdenum, sodium, nickel, phosphorus, platinum, palladium,lead, antimony, selenium, tin, strontium, thallium, titanium, uranium,vanadium, tungsten, yttrium, zinc, scandium, lanthanum, rare earthelements and divalent transition metals.

In another embodiment, the biosorbent element is for method or use inremediation of wastewater having the metals to be removed and the wateris wastewater. In another embodiment, the biosorbent element is formethod or use in remediation of mining process water having the metalsto be removed and the water is mining process water. In anotherembodiment, the wastewater is domestic wastewater, urban wastewater,industrial wastewater or combinations thereof. In a further embodiment,the industrial wastewater comprises effluent from a mining operation.

In another embodiment, the biosorbent element is for method or use inremediation of wastewater having the metals to be removed and the wateris wastewater, optionally before the wastewater contacts activatedcarbon.

In another embodiment, the biosorbent element is for method or use inremediation of mining process water having the metals to be removed andthe water is mining process water. In another embodiment, the wastewateris domestic wastewater, urban wastewater, industrial wastewater orcombinations thereof. In a further embodiment, the industrial wastewatercomprises effluent from a mining operation.

In an embodiment, the biosorbent element contains a dried Euglenabiomass, or a fraction thereof, a wet Euglena biomass, or a fractionthereof, or an exudate of a culture of Euglena, or a fraction thereof,that includes glutathione, metallothioneins, phytochelatins,polyphosphates, polysaccharides, or combinations thereof. In anotherembodiment, the biosorbent element in contained in a dialysis containeror dialysis bag. In another embodiment, the biosorbent element isembedded in diffusive gradient in a plurality of thin films, optionallya diffusion gradient technology (DGT). In another embodiment, the driedEuglena biomass, or a fraction thereof, a wet Euglena biomass, or afraction thereof, or the exudates of a culture of Euglena, or a fractionthereof, is spherical and/or gelatinous. In another embodiment, thebiosorbent element includes an immobilizing matrix to encapsulate thedried Euglena biomass, or a fraction thereof, a wet Euglena biomass, ora fraction thereof, or the exudates of a culture of Euglena, or afraction thereof. In another embodiment, the immobilizing matrixincludes a resin, a polymer plastic, or diffusive gradient in thinfilms. In another embodiment, the immobilizing matrix comprises agar,agarose, alginate, carrageenan, cellulose, chitosan, polystyrene,polyurethane, polyvinyl, or combinations thereof. In a furtherembodiment, the immobilizing matrix contains sodium alginate.

In another embodiment, the fraction is fraction A, B, C or D from AF4fractionation, optionally fraction C. In another embodiment, fraction Ccomprises fraction eluant between 9 and 10 min retention time from AF4fractionation. The skilled person readily recognizes alternates to AF4fractionation to obtain a fraction with biochemical characteristics andmetal binding properties similar to fraction C.

In another embodiment, the biosorbent element is for method or use inremediation of wastewater, optionally mining process water, to removeAu, Ag, Cu, and/or Pb, optionally the wastewater has been depleted ofCN, optionally the biosorbent element comprises unencapsulated Euglenabiomass, encapsulated Euglena biomass beads, or beads comprisingexudates of a culture of Euglena, optionally the Euglena biomass washeat treated at a temperature of at least 40, 45, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120,125, 130, 140, 145, 150, 155 or 160° C., optionally the heat treatmentcomprises heating the biomass for at least 12, 24, 36, 48, 60, 72, 84 or96 hours, optionally the bead comprises bead size of at least 0.1, 0.2,0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mm, optionally thebead comprises bead size of at most 4, 5, 6, 7, 8, 9, or 10 mm,optionally the pH of the beads is at least 2, 3, 4, 5, 6, or 7,optionally the pH of the beads is at most 6, 7, 8, 9, 10, 11, 12 or 13,optionally the beads are housed in a plurality of columns, optionallythe columns arranged to produce a column effluent, optionally theplurality of columns are arranged in a series comprising at least afirst column, a second column and a third column, and the solution flowsthrough the columns sequentially to produce the column effluent,optionally the first column selectively binds to Au, Ag, Cu or Pb, thesecond column selectively binds to Au, Ag, Cu or Pb, and the thirdcolumn selectively binds to Au, Ag, Cu or Pb, optionally before thewastewater contacts activated carbon.

As used in this disclosure, the term “gelificate” and its derivatives,as used herein, is defined as the process of turning a substance into agelatinous form. This process comprises converting liquid substancesinto solids with the help of a gelling agent. Common gelling agents comefrom natural sources and include agar-agar, gelatin, carrageenan, gellangum, pectin and methylcellulose.

In an embodiment, the biosorbent element is used in accord to themethods described herein. In another embodiment, the binding of a targetmetal includes contacting a solution containing a target metal with thebiosorbent element described herein, and optionally separating thecomplex from the solution.

The present disclosure includes a system for binding a target metalcomprising a biosorbent element described herein and activated carbon.

The systems, methods, uses and biosorbents disclosed herein can beapplied to any microorganism. The systems, methods, uses and biosorbentsdisclosed herein are scalable at higher capacity.

It will be appreciated by a person skilled in the art that embodimentsof the uses of the present disclosure can be varied as described hereinfor the methods of the present disclosure.

The following non-limiting Examples are illustrative of the presentdisclosure:

Example 1: Equilibrium and Kinetic Studies of Cu (II) and Ni (II)Biosorption on Non-Living Euglena gracilis I. Introduction

Kinetic and sorption properties of dried Euglena cells were assessed inmono (Cu or Ni) and bi-metallic solutions (Cu+Ni) at metalconcentrations typical of wastewaters (Gopalapillai et al., 2008;Mandavi et al., 2012). Kinetic modeling of metal sorption aids thedesign, optimization and commercial application of algal metal removalprocesses. Kinetic data describes the rate at which metal ions are takenup by the sorbent and therefore determines the residence time requiredfor effective removal.

II. Experimental Procedures A. Test Organism, Medium and CultureConditions

Euglena gracilis Klebs were obtained from Boreal Laboratory Supplies Ltd(St. Catharines, ON, Canada). Non-axenic cultures were grown in mediumconsisting of 0.01 g·L⁻¹ CaCl₂) (Bishop Canada Ltd), 1.0 g·L⁻¹CH₃COONa.3H₂O (Caledon Ltd, Canada), 1.0 g·L⁻¹ “Lab-Lemco”, 2.0 g·L⁻¹tryptone (Oxoid LDD, Basingstoke, Hampshire, England) and 2.0 g·L⁻¹yeast extract (Oxoid LDD, Basingstoke, Hampshire, England). All mediawere prepared using Milli-Q water. The pH of the medium was adjustedusing 1M HCl or NaOH after autoclaving and maintained between pH 3-5 at20° C. in a Conviron (CMP5090) environmental chamber (ControlledEnvironments Ltd., Winnipeg, MB, Canada). E. gracilis were grown under aphotoperiod of 18:6 (light:dark) at an intensity of 210 μmol·s⁻¹. Toobtain non-living Euglena, post-harvest Euglena biomass was cooled to−80° C. (Forma Scientific, USA) for at least 24 h and subsequentlyfreeze-dried (LabConco, USA) for at least 48 h and milled. Glassware wasimmersed in 20% HNO₃ prior to use for at least 24 h and triple-rinsedwith Milli-Q water to avoid metal contamination. In addition, anyglassware used for culture growth was autoclaved to mitigate bacterialcontamination.

B. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

Dried biomass was analysed with attenuated total reflectance (ATR) usingan ATR-FTIR spectrometer (Nicolet 380, Thermo, USA) in the absorbancemode (range: 4500-500 cm⁻¹) with 32 scans at a spatial resolution of 4cm⁻¹.

C. Metal Solutions

Cu (II) and Ni (II) stock solutions (0.01 mol·L⁻¹) were prepared withCuSO₄.5H₂O (Caledon Laboratory Chemicals) and NiSO₄.6H₂O (BDHChemicals), respectively. The pH of working metal solutions was adjustedwith 0.1 mol·L⁻¹ HCl and 0.1 mol·L⁻¹ NaOH (Accumet, XL15, USA). Metalconcentrations were determined utilizing inductively coupled plasma massspectrometry (ICP-MS) (X Series II, ThermoScientific, USA). Rhodium wasused as an internal standard. The accuracy of the ICP-MS measurementswas assessed using SLEW-3, SLR-4, SLR-5, 1-BIS and 5-BIS certifiedreference material (National Research Council, Canada).

D. Cu²⁺ and Ni²⁺ Biosorption

The amount of Cu²⁺ and Ni²⁺ adsorbed at equilibrium, q (μg·g⁻¹) wascalculated with the following equation:

$\begin{matrix}{q = \frac{\left( {C_{i} - C_{eq}} \right)V}{m}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where C_(i) is the initial concentration of the metal ion prior toadsorption (μg·L⁻¹) and C_(eq) is the equilibrium concentration of metalions in the aqueous phase. Vis the volume (L) of the aqueous phase and mis the dry weight mass of the adsorbent (g). Each experiment wasperformed in duplicate and the results are presented as averages. Allbiosorption experiments were performed utilizing the batch technique.

E. Sorption Kinetics

Kinetics experiments were performed in duplicate at a constanttemperature (20° C.) in 50 mL centrifuge tubes (Fisher Scientific)containing E. gracilis (1 g·L⁻¹) suspended in Milli-Q spiked with metalsolutions of either Cu(II) and/or Ni(II). Kinetic studies were conductedat pH 5.0 and agitated at 70 rpm for 240 min. Kinetic studies werecarried out for sorption of Cu²⁺ and Ni²⁺ as a function of contact timeat four initial concentrations for each metal (Cu²+=20 and 50 μg·L⁻¹, 1and 25 mg·L⁻¹; Ni²⁺=1, 2, 4, and 20 mg·L⁻¹) at pH 5 on non-livingEuglena gracilis. Aliquots (7 mL) were removed from solution atpre-determined intervals over the time-course of the experiment, 0.7μm-filtered (GFF, Merck Millipore, Ireland), acidified (ultrapure HNO₃70%) to pH 2.0 and metal concentrations were measured by ICP-MS.Adsorption kinetics models were used to evaluate the overall rate of Cu(II) or Ni (II) removal from Euglena—single metal solutions. Sampleswere taken at time intervals of 0, 10, 20, 30, 60, 90, 120, and 240minutes. Two different, non-linear models were employed:

The pseudo-first-order kinetic equation (PFO; Lagergren, 1898):

q _(t) =q _(e)(1−e ^(−kt))  (Equation 2)

where q_(e) is the amount of metal adsorbed (μg g⁻¹ or mg g⁻¹) atequilibrium, q_(t) is the amount of metal adsorbed (μg g⁻¹ or mg g⁻¹) attime t, k₁ is the PFO equilibrium rate constant (min⁻¹) and t is contacttime (min).The pseudo-second-order kinetic equation (PSO; Ho et al., 1996):

$\begin{matrix}{q_{t} = \frac{q_{e}^{2}k_{2}t}{1 + {q_{e}k_{2}t}}} & \left( {{Equation}\mspace{14mu} 3} \right)\end{matrix}$

where q_(e) and q_(t) are metal adsorbed at equilibrium and time t,respectively, k₂ is the PSO equilibrium rate constant, and t is contacttime.

F. Sorption Equilibria

Sorption of Cu (II) and Ni (II) on living E. gracilis (1 g·L⁻¹) wereexamined in batch adsorption-equilibrium experiments in duplicate (120min) at a constant temperature (20° C.) and agitated at 70 rpm. Blanktrials without Euglena cells and trials without added metal solutionwere performed for each tested metal concentration. The pH levels ofboth mono- and bi-metallic solutions were maintained at 5.0 over theduration of the experiments with additions of 0.1 M HCl and/or 0.1 MNaOH. The effect of metal initial concentration was studied at pH 5.0 inmono-metallic solutions with values ranging from 5 μg·L⁻¹ to 1.5 mg·L⁻¹(Cu) and 5 μg·L⁻¹ to 1 mg·L⁻¹ (Ni). Bi-metallic solutions were preparedwith initial Cu²⁺ and Ni²⁺ concentrations ranging from 10 μg·L⁻¹ to 6mg·L⁻¹ and from 2 μg·L⁻¹ to 200 μg·L⁻¹, respectively. Metalconcentrations were determined using ICP-MS. The Langmuir and Freundlichisotherm models were used to analyze biosorption data.

G. Statistics: t-Tests

Paired t-test analysis was used to evaluate differences in metalsorption between systems with different initial metal concentrations.

III. Results and Discussion A. Characterisation of Dried Euglena Cells

The FTIR spectrum of dried Euglena cells (FIG. 1) showed differentfunctional groups. The intense and strong bands at 3040 and 1640 cm⁻¹were related to C—H and C═C in aromatic hydrocarbons. The stretchingvibrations of O—H (1700 cm⁻¹) and C—O (1380 cm⁻¹) were attributed to thecarboxylic functional group. The C═O in amides was found at 1640-1660cm⁻¹. Overall, dried Euglena cells possessed the main functional groups(e.g. carboxylic, amino and thiol) for metal binding.

B. Sorption Kinetics

Both metals (Cu and Ni) were found to adhere more closely to the PFOmodel (Table 1) congruent with previous non-living biomass studies (Liuet al., 2009; Rao et al., 2005). A strong agreement with the PFO modelsupported, while not wishing to be limited by theory, that the mechanismof adsorption was controlled by the physical attraction of metal ionsonto unoccupied sites on the biomass as opposed to a process ofchemisorption (e.g. agreement to the PSO model) in which metal ionsshare electrons with functional groups on the cell surface (Ho andMcKay, 1998; Plazinski, 2013). Sorption of Cu and Ni to Euglena occurredrelatively quickly within 10 to 30 min (FIG. 2 and FIG. 3) whichindicates an attraction between the metals and the biomass. A strongaffinity between sorbent and sorbate is an integral component of anyapplication of biosorption to the remediation of metal-bearing effluent(Volesky, 2003). Using biological material to achieve metal removalrequires a mass transfer of metal ions to the biomass from solutiondriven by a mutually attractive force (e.g. electrostatic, ionexchange). This affinity between Euglena biomass and metal ions issupported, while not wishing to be limited by theory, by the steepinitial rise of the curves (FIG. 2 and FIG. 3). The PFO kinetic rate(k₁) generally increased as the initial concentration of metals wasincreased (Table 1 and Table 2). Previous biosorption studies havereported similar effects on kinetic constants while others have foundhigher values at lower concentrations (Cordero et al., 2004; Jaikumarand Ramamurthi, 2009). Amounts of metal sorbed in kinetic experimentswere found to increase with initial metal concentration for both Cu andNi (p<0.05). As the initial concentration of Cu increased from 0.02mg·L⁻¹ to 25 mg·L⁻¹, metal loading also increased from 0.0066 mg·g⁻¹ to8.40 mg·g⁻¹. Similarly, as Ni initial concentrations increased from 1 to20 mg·L⁻¹, loading increased from 0.341 mg·g⁻¹ to 3.66 mg·g⁻¹ (Table 1and Table 2). An increase in the initial concentration of Cu (˜1250×)resulted in an approximately proportional increase (˜1270×) in theamount of Cu sorbed to the biomass. In contrast, the amount of Ni sorbedincreased ˜12× concurrent with a 20× increase in initial concentration.A potential reason for this result could be that ligands which possess ahigh affinity for Cu are likely to contain N or S donor atoms (e.g.proteins, amino acids, thiols; FIG. 1) which tend to form stronger bondswith Cu as compared to other groups (Kiefer et al., 1997). Compared toother algal species, Euglena possesses higher proportions of protein andthiol structures which could contribute to the preferential sorption ofCu over Ni.

TABLE 1 Kinetic model parameters for the biosorption of Cu on non-living Euglena gracilis cells - pseudo-first order model. Initialconcentration q

 (±SE) k_(f) r² % removal (±SE) 20 μg/L 6.65 μg/g 0.147 0.725 43 (n = 1)50 μg/L 27.0 μg/g 0.224 0.974 65 (±2) (±0.647) 1 mg/L 0.587 μg/g  0.2870.974 60 (±1) (±0.014) 25 mg/L 8.40 μg/g 1.03 0.981 34 (±5) (±0.157)

indicates data missing or illegible when filed

TABLE 2 Kinetic model parameters for the biosorption of Ni on non-living Euglena gracilis cells - pseudo-first order model. Initialconcentration q

 (±SE) k_(f) r² % removal (±SE) 1 mg/L 0.341 mg/g 0.182 0.913 38 (±7)(±0.016) 2 mg/L 0.406 mg/g 0.339 0.742 22 (±4) (±0.034) 4 mg/L 0.752mg/g 0.410 0.648 8 (±3) (±0.106) 20 mg/L  3.66 mg/g 0.175 0.840 13 (±8)(±0.242)

indicates data missing or illegible when filed

C. Sorption Equilibria

In this disclosure, the biosorption of Cu²⁺ and Ni²⁺ was evaluated atindustrially relevant (e.g. wastewater which has undergone primarytreatment) concentrations (e.g. ≤25 mg·L⁻¹). The biosorption of bothCu²⁺ and N²⁺ in single-metal solutions increased with increasingequilibrium concentration (FIG. 3 and FIG. 4). The degree of fit (r²) toboth the Langmuir and Freundlich models indicated that the sorption ofCu (0.995 and 0.985, respectively) and Ni (0.996 and 0.985,respectively) by non-living Euglena gracilis can be describedappropriately by either model (p<0.001; both metals and models; Table 3,Table 4, FIG. 4 and FIG. 5). However, the higher r² for the Langmuirmodel compared to Freundlich indicated that metal ions were beingadsorbed in a monolayer with functional groups on the surface of thebiomass (Rao et al., 2005). The comparatively high applicability of theFreundlich model supports that sorption also may occur on heterogeneoussurfaces on the cell. Comparable Langmuir parameters (k and q_(max))were found for both metals (p>0.05; Table 3). The maximum capacity forbiosorption was 89.6 μg/g and 34.1 μg/g for Cu and Ni respectively.Despite differences in metal concentrations, Langmuir parameters (e.g.q_(max) and b) were analogous to previous studies (Chen et al., 2008;Rao et al., 2005). The values of k ranged from 556 to 625 for Ni and Cu,respectively, with no significant differences between metals (p>0.05)(Table 3).

TABLE 3 Langmuir adsorption isotherm parameters for the biosorption ofCu and Ni on non-living Euglena gracilis cells at pH 5. Metal k q

 μg g

r² % Removal Cu 625 89.6 (83.1-99.2) 0.995 49 (±12) Ni 556 54(31.3-37.1) 0.996 32 (±7)

indicates data missing or illegible when filed

TABLE 4 Freundlich adsorption isotherm parameters for the biosorption ofCu and Ni on non-living Euglena gracilis cells at pH 5. Metal K_(f)

/n r² % Removal Cu 0.502 0.697 0.985 49 (±12) Ni 0.196 0.770 0.989 32(±7)

indicates data missing or illegible when filed

The biosorption of Cu and Ni was also examined in binary metal solutionsto better emulate the mixed composition of industrial wastewaters (FIG.6). Sorption was assessed as a function of the ratio of the initialconcentrations of Cu to Ni. When compared to single-metal solutions, Cuand Ni total metal uptake did not differ significantly in terms ofsorption capacity as a function of initial metal concentrations(p>0.05). At Cu:Ni<1, Ni sorption was greater than Cu (p<0.05; FIG. 6)whereas at Cu:Ni>1Cu sorption was higher (p<0.05). This contrasts withlive Euglena cells wherein Cu and Ni uptake was similar between Cu:Niratios (p>0.05) (Winters et al., 2016). Ni sorption was supressed athigh concentrations of Cu (Cu:Ni>1) whereas the opposite occurred atrelatively higher Ni levels, which supports, while not wishing to belimited by theory, an antagonistic biosorption competition between Cuand Ni ions for commonly shared binding sites. The equilibrium uptake(q_(e)) of Ni decreased with the increasing quantity of Cu ions. Theantagonistic effect of Cu ions on the equilibrium Ni uptake was dominantat higher initial Cu concentrations.

IV. Conclusions

This Example assessed metal sorption at typical concentrations (<100mg·L⁻¹) found in industrial wastewaters after a primary form oftreatment. To our knowledge, this is the first instance in which Euglenahas been studied for this purpose. Non-living Euglena gracilis has beenshown to demonstrate comparable biosorption capacities (34-89 μg g⁻¹) asother eukaryotic organisms in the removal of Cu and Ni from aqueoussolutions. FTIR analysis indicated the presence of functional groups(e.g. carboxyl, amide) which have been previously identified asimportant metal binding sites for metal biosorption. Sorption kineticsfollowed the PFO model supporting, while not wishing to be limited bytheory, a physically-based form of biosorption. Both the kinetic rate(k₁) and the amount of metal sorbed were found to rise with increasinginitial concentration. Although sorption occurred rapidly (10-30 min),Cu was more efficiently taken out of solution than Ni. In single-metalsolutions both Cu and Ni were found to exhibit a greater degree of fitto the Langmuir model which supports, while not wishing to be limited bytheory, that metal ions bind to uniform sites on the cell surface in amonolayer. Similarly to kinetic results, Cu sorption was found to begreater in single-metal solutions than Ni although Euglena exhibited asimilar affinity for both metals. Langmuir parameters were found to becomparable to sorption of Cu and Ni by similar organisms. Inbinary-metal systems, sorption of Cu was supressed at relatively highconcentrations of Ni and conversely, Ni sorption was inhibited at higher(e.g. >10) Cu:Ni ratios. As to be shown next in Example 2, the removalpotential of non-living Euglena, whether dried or wet, was conductedusing actual multiple metals-containing water from a mining operation.

Example 2A: Multiple Metals Bind to Spherificated Wet Euglena Biomass,Dried Euglena Biomass and Euglena Exudates I. Introduction

The recovery of gold from water in mining operation is of significantinterest to the mining industry. Mining operation often utilizes acarbon filtration system to recover gold in their process. An issue withthis method is the reduced efficiency of the carbon filter by thepresence of copper, which is preferentially bound by the filter, therebyreducing the recovery of gold from the water. The strategy employed byin this study was to use the Euglena-based platform to remove the copperfrom the water prior to carbon filtration, thereby improving the yieldof gold via carbon filtration.

Several different forms of the Euglena platform were tested for theirability to bind heavy metals from mining process water samples. Themining process is an open pit CN leaching operation. The mining processwater and in this Example is between pH of 9-11, and contains between100-800 ppm total CN. Euglena gracilis has the ability to secretedissolved organic materials in response to heavy metal stress. Thesedissolved organic materials are constituents of the exudates that canpotentially bind and de-toxify the heavy metals in solution.Spherification of Euglena or exudates of Euglena provides a greater andmore concentrated surface area for metal binding and also provides amethod for the removal of the Euglena and the bound metals after theincubation period. In this Example, both live and non-living Euglenawere tested for their ability to remove metals by incubating the cellswithin the mining process water. Further, spherificated Euglena-basedplatform including spherificated exudates of Euglena were also testedfor their ability to bind metals.

II. Experimental Procedures A. Preparation of Wet Euglena Biomass, DriedEuglena Biomass and Exudates of a Culture of Euglena

Euglena gracilis was grown in media and harvested by solid liquidseparation using centrifugation, where the wet solid biomass wasretained, and the media discarded. Dried Euglena cells were obtained bydrying the biomass.

For the collection of exudates, instead of discarding the media asabove, the exudates were recovered from media taken from the Euglenaculture. 5 mL of media was used to obtain 50 mg of dry weight of mediacontaining the exudates.

B. Spherification Process

The spherification process involves using a sodium alginate solution toencapsulate the Euglena biomass and the exudates of Euglena. Thisprovides a greater and more concentrated surface area for metal bindingand also provides a method for the removal of the Euglena and the boundmetals after the incubation period. The “Sphered” wet Euglena biomass,dried Euglena biomass, as well as exudates, i.e. spent media thatEuglena grew in, were spherificated in this study. The “Free” wetEuglena biomass, dried Euglena biomass, and Euglena exudates arematerials that have not been spherificated.

The spherification process involves mixing media containing wet Euglenabiomass, dried Euglena biomass, or exudates of a culture of Euglena withan equal volume of 4 g·L⁻¹ sodium alginate solution. This mixture isdripped into a gently stirring solution of chilled 2 g·L⁻¹ CaCl₂). Uponcontact with the CaCl₂) solution, the droplets form spheres. Once themixture being encapsulated has been exhausted and the spheres haveformed, the spherificated materials are poured through a strainer andstored in a closed vessel until used.

C. Metals Binding

The “Free” Euglena treatments were prepared by using 1.5 g (dry weight)sample of either wet Euglena biomass, dried Euglena biomass, or exudatesof a culture of Euglena. This quantity was then mixed with the miningprocess water sample for 24 hours after which the sample was pouredthrough a 5 μm filter and submitted for inductively-coupled plasmaoptical emission spectrometry (ICP-OES) analysis.

The “Sphered” Euglena treatments were prepared by using 1.5 g (dryweight) sample of either wet Euglena biomass or dried Euglena biomass,which was spherificated as described above. For the Sphered exudates, 50mg of media containing the exudates (dry weight) in which the Euglenahad been growing in was spherificated using sodium alginate via thestandard procedure for spherification as described above. The spheresproduced were mixed with the processing mining water sample for 24hours, after which the sample was poured through a 5 μm filter andsubmitted for ICP-OES analysis.

D. Preparation and Determination of Metals Complexed by Euglena andEuglena Exudates

Preparation and determination of multiple metals in samples treated withthe Euglena platform were carried out at the Lakefield Laboratory of SGSMinerals Services Geochemistry (SGS). Samples in aqueous CN processsolutions were acidified with HCL, HNO₃ and H₂O₂, and then diluted into20% hydrochloric acid. Diluted acidified solutions were analyzed by theVarian Vista ICP-OES system. Quality controls included one method blank,one sample replicate and two calibration check solutions analyzed withevery batch of 24 samples or less.

E. Preparation and Determination of Au Complexed by Euglena and EuglenaExudates

Preparation and determination of gold in samples treated with theEuglena platform were carried out at the Lakefield Laboratory of SGSMinerals Services Geochemistry. Gold present in a solution sample of CNor acid based matrix, plus an inquart of silver nitrate are fire assayedusing Pb flux and cupelled to produce a doré bead. The bead wasdissolved using HCl and HNO₃ and the resulting solution was submittedfor analysis. Gold content was analyzed by flame atomic absorptionspectrometry (AAS) using acid matrix matched calibration materials.Quality controls included one method blank per 7 samples; 1 duplicateper 7 samples; 1 sample spike taken through the digestion per batch; andcalibration materials that cover the linear range.

III. Results and Discussion A. Binding of Multiple Metals bySpherificated Euglena Exudates

The results in Table 5 show amounts of various metals in samples treatedwith different forms of the Euglena platform compared to the untreatedwater (Control 1 & 2). Values are showing the amount of metals remain insolution after treatment. On average the level of gold in the controlsamples was 0.136 mg·L⁻¹. A promising result with this platform was thatthe gold concentration was reduced by only 6% across all variants of theEuglena platform (Avg. 0.128 mg·L⁻¹). In contrast, the levels of copperare markedly reduced compared to the controls, regardless of theplatform variant used. On average the Euglena treatments removed ˜82% ofthe copper from the water samples. The high copper binding capacity ofvarious forms of the Euglena platform in process water obtained from anactual mining operation which contains multiple metals shows a practicalapplication of the Euglena platform described herein.

TABLE 5 Multiple metals binding to spherificated wet Euglena biomass,dried Euglena biomass, and exudates of a culture of Euglena gracilis.Sample Sphered Free Sphered Free Element Control Control Euglena EuglenaEuglena Euglena Sphered (ppm) (1) (2) (Wet) (Wet) (Dried) (Dried)Exudates Au 0.134 0.138 0.127 0.129 0.13 0.13 0.129 Ag 0.0527 0.05010.073 0.124 0.0499 0.0609 0.184 Al 3.53 3.35 0.08 0.004 0.024 0.0250.034 As 0.0158 0.0154 0.0031 0.0031 0.0025 0.003 0.0038 Ba 0.03120.0296 0.00862 0.0137 0.112 0.0739 0.00781 Be 0.000115 0.000107<0.000007 <0.000007 <0.000007 <0.000007 <0.000007 B 0.333 0.345 0.3410.349 0.376 0.369 0.552 Cd 0.0167 0.0151 0.000879 0.000668 0.0004480.000506 0.000812 Cr 0.00525 0.00533 0.00195 0.00103 0.00096 0.000990.00181 Cu 223 208 37.7 39.2 35.1 40.8 31.5 Fe 4.74 4.68 0.126 0.010.008 0.014 0.055 Mg 37.6 38.2 35.6 49.9 36.1 38.2 61.9 Mn 0.33 0.3060.0135 0.00679 0.00161 0.00462 0.0192 Mo 1.83 1.86 1.77 1.83 1.71 1.771.68 Na 2060 2040 1970 2060 1980 2040 2010 Ni 0.155 0.16 0.161 0.1670.16 0.148 0.143 Pb 0.0442 0.0384 0.00112 0.00005 0.00021 0.000160.00074 Se 1.31 1.27 0.833 0.86 0.818 0.823 0.821 Sr 0.665 0.667 1.620.631 0.864 0.598 11.4

A key performance metric of the different treatments is to compare theRatio of the Weight of the Active Material to Weight of Copper Removed(Table 6). Both “Free” wet Euglena biomass or dried Euglena biomass, aswell as the “Sphered” wet Euglena biomass or dried Euglena biomassshowed similar relationships, with ratios in the range of 53.2:1 to54.9:1.

TABLE 6 Weights of Active Materials and Effectiveness in Removing Metalsby Free and Sphered wet Euglena biomass, dried Euglena biomass andexudates (media) of a culture of Euglena gracilis. Initial Weight ofCopper Weight of Copper in Final Weight of Ratio of Weight of RemovedActive 150 mL of Weight of Removed Active Material to From MaterialSolution Copper Copper Weight of Copper Solution Treatment (mg) (mg)(mg) (mg) Removed (%) Free Euglena (Wet) 1500 33.5 5.9 27.6 54.4:1 82%Free Euglena (Dry) 1500 33.5 6.1 27.3 54.9:1 82% Sphered Euglena (Wet)1500 33.5 5.7 27.8 54.0:1 83% Sphered Euglena (Dry) 1500 33.5 5.3 28.253.2:1 84% Sphered Media 50 33.5 4.7 28.7  1.7:1 86%

The most successful treatment with respect to this ratio was the Spheredexudates (labeled as “Sphered Media” in Table 6), which showed a ratioof 1.7:1. This result demonstrated that the Sphered exudates performed˜31× better than Free wet or dried Euglena biomass, as well as Spheredwet or dried Euglena biomass.

All of the treatments (Table 6) showed removal rates of 82% to 84% (ofcopper), which supports, while not wishing to be limited by theory, thespherification process has a minimal impact on the removal of the copper(and other metals according to Table 5).

Other metals and metalloids of note that have been removed are aluminum(˜98%), arsenic (˜80%), cadmium (˜95%), chromium (˜75%), iron (>99%),manganese (˜96%), lead (>99%) and selenium (˜35%).

IV. Conclusions

Using “Free” wet or dried Euglena biomass or “Sphered” wet or driedEuglena biomass to capture copper from process water sample gives an82-84% removal of copper. The ratio of the Weight of Active Material tothe Weight of Copper Removed is between 53.2:1 and 54.9:1.

Sphered exudates removes 86% of the copper in the process water sample,and is useful in a 1.7:1 ratio of Weight of Active Material to Weight ofCopper Removed; this is ˜31× better than when compared to either theFree Euglena biomass or the Sphered Euglena biomass ratios.

Metal binding capacities of free and sphered Euglena exudates arecomparable. The main benefit of using the sphered Euglena exudates isthe ease of removal of complexes after metals have bound to thematerials. To recover the metal-bound complexes, handlers are notrequired to centrifuge the samples post-treatment. This saves time,energy and reduces the risk for the accidental release of the capturedmetals during sample handling.

Example 2B: Selective Metals Binding to Euglena Exudates and EuglenaBiomass Subjected to Different Post-Harvest Treatments Prior toSpherification I. Introduction

As shown above in Example 2A, spherification of Euglena provides certainadvantages in metals recovery. Next, the functionality of differentlygenerated beads as a binding agent for Au, Ag, Pb and Cu in threescenarios is disclosed. In the first scenario, tests determined ifcolumns containing Euglena-beads could operate downstream of carboncolumn or in the excess pond in order to capture the Au that passedthrough the carbon column. The second scenario tested whether thecolumns containing Euglena-beads could improve the selective Au bindingproperties of the carbon columns by functioning upstream to remove Cuand Pb. Finally, tests explored whether the columns containingEuglena-beads could function as the primary Au recovery method andcompletely replace the carbon column. Ten variations of beads wereprepared at various drying, heating and pH levels, loaded into columns,and tested. In total, over fifty trials were conducted at differentsites.

Post-harvest treatments prior to spherification optionally affectbinding capacity of Euglena biomass or exudates of a culture of Euglena.In the present disclosure, different post-harvest treatments of Euglenabiomass are found to affect the capacity of active materials in bindingto different metals and the degrees of removal of different metals.

II. Experimental Procedures A. Site Selection and Sample Collection

Eleven different sites (A to K) throughout a mining operation wereselected to represent all possible water chemistries existing on themining operation (FIG. 7). The variety of samples were chosen todetermine how various water chemistries affected bead performance.

B. Bead Preparation

The biochemicals used in this study were derived from Euglena gracilisbased on standard fermentation and utilizes sugar as a carbon source. Toallow for ultimate flexibility and co-production with other materials atscale-up, no “directed expression” techniques were used to achievebiochemical selectivity. Instead, treatment of the biochemical afterextraction was attempted.

Biochemicals were derived either from the insoluble fraction of thebiomass or from their soluble secreted materials (i.e. exudates). Thesematerials were isolated and then processed with various techniques, toattempt to create material with preferential binding to a specificmetal. Sodium alginate was selected to create a matrix that would holdthe biochemicals when binding metals.

For beads made with harvested Euglena cells, an equivalent weight of 100g (dried material) of the Euglena biomass, 100 g of heated material isbrought to 1 L with deionized water (DI) water. This mixture is blendedthoroughly to which 6 g of sodium alginate is added. This mixture isonce again blended thoroughly for a period of 5 minutes or more.

The resulting mixture is then dripped into 2 L of a 4 g·L⁻¹ chilledCaCl₂ solution with stirring. Upon contact with the CaCl₂ solution, thebeads are formed. Once all of the slurry has been dripped into the CaCl₂mixture, the resulting beads are strained of excess solution and thenstored in a sealed container.

For beads made with exudates of a culture of Euglena cells, anequivalent weight of 2.4 g dried material contained in 1 L of liquid isblended thoroughly with 6 g of sodium alginate for 5 minutes or more.This mixture is then dripped into a CaCl₂ solution as described above toform beads. To concentrate the exudates of Euglena, liquid containingthe exudates is evaporated off in a rotovap at 70° C. until the volumehas been reduced by 2.5×. For beads made with this concentratedexudates, an equivalent weight of 6 g dried material contained in 1 L ofliquid is blended thoroughly with 6 g of sodium alginate for 5 minutesor more. This mixture is then dripped into a CaCl₂ solution as describedabove to form beads.

Alternatively, either of the mixtures from above is envisioned to bespherificated, gelificated, encapsulated or immobilized with materialthat will allow the diffusion of metal barring solutions through themwhile retaining the material to be encapsulated. The desired form of theencapsulated material is spherical to maximize surface area of the saidbeads.

In total, ten bead variations were tested to determine efficacy and ifpreferential selectivity to any of the metals was exhibited (Table 7).Euglena biomass (EE) was not encapsulated and was used as a control.Bead size (3 mm and 5 mm) was also varied to see if surface area had aneffect on binding chemistry. Exudates of a culture of Euglena cells wereencapsulated to examine concentration and the effect of increasedsurface area on the performance of the technology. Biomass derivedbiochemicals were then dried (50° C.) and heat-treated (80° C., 120° C.)to explore if this influenced the binding preference to Au (similar toactivated carbon). Biomass derived biochemicals were also prepared atvarying pH (4, 6, 7) to explore the effect of pH on binding chemistry.

TABLE 7 Trial conditions and variables. Bead Temperature NameDescription of Material Bead Size pH Treatment WBS Wet BiomassBiochemicals 3 mm 6 — WB4 Wet Biomass Biochemicals 5 mm 4 — WB6 WetBiomass Biochemicals 5 mm 6 — WB7 Wet Biomass Biochemicals 5 mm 7 — DB50Dry Biomass Biochemicals 3 mm 6 50° C. DB80 Dry Biomass Biochemicals 3mm 6 80° C. DB120 Dry Biomass Biochemicals 3 mm 6 120° C. SB SecretedBiochemicals 5 mm 6 — CSBS Concentrated Secreted 3 mm 6 — Biochemicals(Exudates) CSBL Concentrated Secreted 5 mm 6 — Biochemicals (Exudates)EE Euglena not Encapsulated — — —

C. Standard Column Operation

The standard single column operation was performed by loadingapproximately 50 mL of the bead material into a column (FIG. 8A).Deionized water was pumped through the column to wash the bead material.The column was positioned so that the sample traveled up from the bottomof the column and out through the top. The sample was pumped through thecolumn at a rate of 1 bed volume (BV) per 5 minutes. At each 5-minuteinterval, a sample was taken to compare how the composition of the waterchanged from the beginning of the process. The column was run for 3 BVto test total percentage removal.

The single column operation was performed with bead type WBS at allsites to test how the water chemistry affected bead performance; allbead types were also tested at site K. Selected beads (WBS, WB6, DB50,DB120, SB) were tested at sites A and F to explore the effect of beadtype on performance. These sites were upstream and downstream of thecarbon columns and had diverse metal and CN concentrations. Activatedcarbon was tested at site A and site K, with and without un-encapsulatedEuglena as a pretreatment.

D. CN Destruction and pH Adjustment

A sample of process water was treated with an excess of hydrogenperoxide (H₂O₂) to facilitate the destruction of CN in the sample. Sincemetal ions might precipitate from the solution, a small amount of acidwas added to keep the pH above 4.

The process water was initially tested in its unaltered state, and thensecondly with the addition of H₂O₂, and finally with a constant pH of 4.These tests were done to determine how to maximize column performance.By maintaining a pH of 4, it could be determined whether the pH had apositive effect on the column.

E. Column Series Operation

A column series operation works in the same manner as a single columnoperation, however, two or more columns are connected in a series, witheach column containing a different type of bead, or in anothervariation, a column of activated carbon is included in the series (FIG.8B). The system is operated in the same way as the standard columnoperation, except that the first BV is taken after multiplying thenumber of columns being used by 5 (5 minutes for each column to processa BV). For instance, if 3 columns are connected in a series, the systemmust run for 15 minutes to collect a sample that has passed through thewhole system.

Multiple series were tested with different bead and/or carboncombinations for sites A and K. The beads were collected to examine thedistribution of metal in the series. The objective was to determine ifthe series could either increase the performance of the carbon column,or replace it.

F. Water Analysis

The process water sample was tested with an Atomic AbsorptionSpectrophotometer (AAS) before it was introduced to the column(s) todetermine the concentrations of the metals of interest. After each bedvolume of pond sample was passed through the column, a sample wascollected and again tested using the AAS. The difference between theinitial concentration and the concentration after processing each bedvolume of each sample was calculated and expressed as a percentageremoval. When multiple bed volumes of sample were processed, the averagepercentage removal was reported.

G. Assaying of Metal on Beads

The bead material from the experiments was collected and sent to SGS foranalysis. SGS dried the beads and then dissolved them in aqua regia (avery strong acid). The resulting solution was analyzed on the AAS thatgave the concentration of metal per kg of dry bead. This was done totest the loading and selectivity of the bead columns and carbon columns.

III. Results and Discussion A. Site Selection and Analysis

Samples were taken from eleven different sites throughout the miningoperation to determine how various water chemistries impacted the beadperformance (FIG. 7). As expected, each site possessed varyingconcentrations of Au, Ag, Cu and Pb, as well as varying pH and CN levels(Table 8).

TABLE 8 Chemical compositions of samples. Concentration (ppm) Site Au AgCu Pb Free CN Total CN pH A 1.03 0.53 374 15.6 90 334 11.9 B 0.48 1.19500 8.95 170 552 11.9 C 0.59 0.89 432 0.28 130 462 11.0 D 0.71 0.94 45912.7 500 711 11.9 E 0.34 0.82 431 0.55 620 717 11.1 F 0.16 0.54 440 7.68570 674 11.9 G 0.15 0.55 428 0.36 530 717 11.1 H 0.35 0.72 360 0.91 250472 10.1 I 0.27 0.39 323 0.42 140 361 9.8 J 0.27 0.54 244 0.18 0 109 9.1K 0.25 0.49 242 0.14 0 133 9.1

The highest concentrations of Au and Ag were observed directly off theleach pads (A, B, C) and prior to the column trains (D, E), while thelowest concentrations were observed after the carbon trains (F, G).There was a slight increase in concentration at the pregnant pond (H),but again lower amounts by the excess ponds (I, J, K).

The concentration of both Cu and Pb significantly decreased from thefirst stages (A, B, C) to the end (J, K) of the process. The samephenomenon was observed with pH levels, as they tended to decrease frombeginning to end (starting as high as 11.9 pH and dropping to a low of9.1).

The amount of total CN and free CN behaved differently. Both wereobserved to be at mid-range at the beginning of the process, butsignificantly increased in concentration at the pregnant pond (afteradditional CN was added to the process). The amount of total and free CNgradually decreased to the lowest levels at sites J and K.

Site K had low levels of both CN and metals to allow testing of beadperformance in a potentially more challenging environment. Samples fromsite K was selected in particular as the testing ground to determinewhether the surface area, biochemical content/type, heat treatment andpH of the bead would have an effect on the performance of the beads.

B. Column Operation: Bead Optimization

In total, 23 trials were conducted to examine how the various beadsreacted at the different sites (Table 9).

TABLE 9 Bead type optimization for selected sites. Removal (%) Bead TypeAu Bead Type Au Bead Type Site K WBS 28% 70% 35% — WB4 16% 27% 22% — WB618% 21% 14% — WB7 22% 23% 22% — DB50 22% 11% 29% — DB80 18% 37% 25% —DB120 24% 19% 29% — SB 22% 34% 23% — CSBS 28% 29% 39% — CSBL 14%  5% 23%— EE 24% 35%  8% 14% Site A WBS 36% 77% 26% 95% WB6 37% 47% 27% 96% DB5023% 18% 16% 94% DB120 45% 47% 46% 75% SB 36% 45% 26% 96% EE 17% 30%  3%97% Site F WBS 31% 39% 19% 85% WB6 19%  9%  7% 71% DB50 22% 19% 19% 95%DB120 16% 19% 10% 56% SB 25% 19% 11% 69% EE 31%  9%  7% 86% †Some valuesnot reported due to problems with low initial values and assayprecision.

When all beads were tested at site K, results showed that bead WBS (3mm) demonstrated higher Au, Ag and Cu removal percentages when comparedto bead WB6 (5 mm), demonstrating that a greater surface area providedhigher binding capabilities. This disparity was even greater when small(CSBS) and large (CSBL) concentrated secreted biochemical (i.e.exudates) beads were compared.

When secreted and biomass derived biochemical beads were contrasted, nosignificant trends were observed. Furthermore, when the secretedbiochemicals (exudates) were concentrated in the bead (SB vs. CSBL)metal removal did not increase as predicted.

Another variable analyzed was the heat-treating technique ofbiomass-derived biochemicals. For this variable, beads WBS (wet), DB50(50° C.), DB80 (80° C.) and DB120 (120° C.) were compared. Bead WBSshowed an overall greater percentage removal of all metals. Bead DB120did show a higher affinity for Au when compared to bead DB80, whichcould be due to the heat treatment method that was similar to activatedcarbon. Ag removal also decreased with further heat treatment of thebeads.

Lastly, it was investigated if beads prepared at different pH levelsduring the encapsulation process had an effect on their metal bindingability. Beads WB4, WB6 and WB7, with respective pH levels of 4, 6 and7, with a control pH of 6 were compared. The results demonstrate thatoverall, bead WB7 had the best overall metal removal in comparison tobeads WB4 and WB6; with the exception of Ag removal, which was higherwith bead WB4.

Additional studies were carried out at sites A and F to further explorethe effect of surface area, biochemical type and heat treatment methodon the performance of the beads. Site A, which was located directly offthe Leach Pad process, had the highest concentration of metals and oneof the lowest levels of CN. While without wishing to be limited bytheory, these conditions are favourable to achieve the optimalperformance for the beads. Conversely, site F, possessed both lowermetal concentrations and higher levels of CN. These conditions wereexpected to unfavourably affect the performance of the beads.

When the surface area of the beads was compared at sites A and F, thesame positive effect of smaller sized beads demonstrated at site K wasobserved. When comparing secreted and biomass derived biochemical beadsat sites A, K and F, the beads had similar performances for each metalexcept Ag; which was consistently removed at a higher percentage bybiomass bead WBS.

When comparing heat treatment techniques at different sites the trendswere very different based on bead type and site. At site A, bead DB120had the highest removal of Au and Cu; while at site K there were nosignificant differences. The Au loading of bead DB120 at site Aexhibited a significant increase compared to all other beads at anyother site. This increase in loading could be due to either the higherAu concentration or the lower CN levels. At site F, the WBS bead had thebest removal of Au. This could demonstrate the importance of the bindingpreference of the bead type and water chemistry of the site. At allsites, the WBS bead had the highest Ag removal.

This series of trials demonstrated the significance that bead type andpreparation method had on performance and selectivity. Bead DB120 wasselected as the candidate for Au binding when used at the beginning ofthe process (site A). There was also a high removal of Cu and Pb withthis bead so pretreatment to remove these metals would be required toachieve selectivity. Bead DB50 was selected as the candidate for Curemoval and bead SB as the candidate for Pb removal because ofrelatively low removal ratios of Au compared to target metals. Thesepromising results were used for optimizing the setup of the remainingtrials.

C. Column Operation: Site Optimization

Site optimization was carried out using bead WBS (Table 10). This beadwas selected for further testing at each sites within the process tolook for trends in performance or selectivity based on site. Bead WBShad the best general performance when tested at sites A, F and K in thebead optimization studies and was the first optimized bead that wasextensively tested in the lab during initial trials. The lab workfocused on optimizing total percentage removal for all metals so this ispotentially why it generally performed better than other beads withrespect to metal removal. WBS was next tested on how it would perform atdifferent sites.

TABLE 10 Performance optimization for all sites for bead WBS. Removal(%) Site Au Ag Cu Pb† A 36% 77% 26% 95% B 46% 66% 29% 93% C 28% — 20% —D 42% 40% 21% 89% E 53% 44% 22% 34% F 31% 39% 19% 85% G 33% 38% 16% — H41% 48% 19% 25% I 22% 54% 18%  3% J 52% 84% 35% — K 28% 70% 35% — †Somevalues not reported due to problems with low initial values and assayprecision.

The highest removal of Au was at site E (53%), site J for Ag (84%),sites J and K for Cu (35%) and site A for Pb (95%). It was observed thatthere was higher removal percentages for Cu and Ag for samples with thelowest level of free CN (sites J and K). Additionally, it was observedthat sites with high total CN levels exhibited poor Cu and Ag removal(site G).

Overall, there were significant bead performance differences based onsite location. These differences could be linked to water chemistry ormetal concentration, or a combination of other factors. The results ofsite optimization demonstrate the importance of not only optimizing beadtype, but also the specific sites within the process. In order to limitthe factors affecting bead performance, an attempt to isolate the effectof CN and pH was made with water chemistry optimization trials.

D. Optimizing Water Chemistry

In both the bead and site optimization trials, general trends linked tolower performance when higher CN levels are present and conversely,higher performance in lower pH conditions. This series of trials had thegoal of isolating the effect of these parameters on bead performance.

To isolate the effect of CN and pH on the performance of the beads, CNwas destroyed and the pH was adjusted in samples from sites A and K.Hydrogen peroxide (H₂O₂) was used to reduce CN levels for sites A and Kto almost 0 ppm from 334 and 133 ppm (total) and 0 and 90 ppm (free),respectively. The pH was also adjusted from 11.9 and 9.1 to 4 for sitesA and K. The performance of the beads was then compared for untreatedwater, CN reduced water and pH lowered, CN reduced water.

TABLE 11 Comparison of unencapsulated Euglena to activated carbon ontheir own and in series. Loading of Metal (g/t) Material Used Au Ag CuPb Site A Carbon 0.22 1.8 800 48 EE* 1.08 0.96 60 90.3 EE + Carbon 3.11.4 750 11 Site K Carbon 0.02 1.4 850 0.17 EE* 0.36 1.02 120 0.02 EE +Carbon 0.64 0.98 840 0.33 *Calculated metal loading based on percentremoval.

At both sites, there were a significant positive correlation todestroying the CN and bead performance (FIG. 9). At site A, combining CNdestruction and pH adjustment significantly improved Ag and Cu removal.Au removal improved slightly when combining CN destruction and pHadjustment. At site K, CN destruction alone had the best effect on Au,Cu and Pb removal; while Ag removal benefited from both CN destructionwith and without pH adjustment. Overall, the reduction of CN (eitherwith or without pH adjustment) improved removal of all metals. This isanother example of how significantly the CN speciation at each site canaffect performance.

The process water being treated is a CN containing solution; the metalions of interest are most likely in a metal CN anionic complex. Whilenot wishing to be limited by theory, it is thought that the beadmechanism of action involves the interaction of metal cations with thebead's material. Having the metal in an anionic complex state couldpotentially impede the performance of the bead material. These trialswere able to demonstrate that destroying CN had a positive effect on theperformance of the beads. Most gold mines around the world are requiredby law to destroy CN and metal CN complexes in their tailings prior todischarge so there is a possibility of locating the beads after thistreatment. The more cost effective method would be to select siteswithin the process with the lowest CN level for optimal performance.

E. Carbon Column Pretreatment

A part of the mining operation involves carbon columns which are presentin the process to selectively adsorb Au/Ag from process solutions. Sincecarbon also has a high affinity for other metals such as Cu, testing howa pretreatment step can increase the selectivity and loading of thecarbon column was crucial. For these experiments, columns were filledwith activated carbon and loading capabilities were compared withpretreated and non-pretreated process solutions.

This technique used unencapsulated Euglena (EE) as a pretreatment priorto the carbon column. Promising results were found with this approachand were demonstrated at sites A and K (Table 11). The loading of Auincreased dramatically, at site A the loading increased by 15 times andat site K it increased over 30 times (FIG. 10). At sites A and K therewas a negligible difference in Cu loading showing a higher selectivityfor Au. At both sites the loading of Ag also decreased, as did Pbloading at site A. Overall, pretreatment with EE increased the Auselectivity of carbon while loading of the other metals either decreasedor remained unchanged (with the exception of Pb at site K).

F. Column Series: Carbon Optimization

Preliminary results indicate that some beads have a higher affinity forcertain metals, supporting that rather than only processing samplesthrough a column containing one bead, it would be beneficial to passsolution through columns in series to selectively remove target metals.The goal of this study was to explore the use of these beads upstream ofa carbon filter to remove metals that were decreasing Au selectivity andenhance carbon columns.

Beads DB50 and SB were selected for these trials based on the results ofthe bead optimization work. Bead DB50 was selected based on its loweraffinity for Au compared to bead WBS and its relatively similar removalof Cu, this bead would have a target of removing Cu in the series. BeadSB was selected because it had the highest preference for Pb removal,therefore this bead would have the target of removing Pb in the series.For both sites A and K, two combinations of beads were used, beads DB50then SB followed by carbon (Column Series 1), or bead SB followed bycarbon (Column Series 2) (Table 12).

TABLE 12 Comparison of Column Series 1 and 2 at site A. Site A Au Ag CuPb Bead g/t % dist. g/t % dist. g/t % dist. g/t % dist. Carbon 0.22 —1.8 — 800 — 48 — Column DB50 16 90 3.9 59 7300 84 980 99.24 Series 1 SB1.7 9 2.4 36 830 10 5.9 0.59 Carbon 0.15 1 0.36 5 600 7 1.6 0.16 ColumnSB 1.7 38 3.8 80 1200 63 110 92 Series 2 Carbon 2.8 62 0.94 20 700 37 108

At site A, the Column Series 1 pre-carbon beads had very significantremovals of their target metals: Cu and Pb, and resulted in loweramounts of Cu and Pb on the carbon column when in series than alone.Though not specifically chosen for, this was also the case with Agbinding, increased removal by the beads resulted in lower amounts on thecarbon. However, it also demonstrated a higher than anticipated removalof Au. While without wishing to be limited by theory, because thesetests used a set volume of process solution, the capacity of the beadswas not met. This could mean that there is an initial higher affinityfor Au in bead DB50 than for carbon. One of skill in the art can readilyrecognize that, once saturated, most of the Au would end up on thecarbon column.

Column Series 2 at site A had very promising results. The first columnwith bead SB significantly removed Ag, Cu and Pb and decreased the levelof these metals on the carbon. This resulted in higher Au loading on thecarbon compared to carbon on its own (over 10 times). When comparing thetwo Series, bead SB produced very similar Au loading results. Thispotentially demonstrates that bead SB has a relatively low threshold forAu loading. A low Au threshold coupled with its demonstrated ability toremove Cu and Pb supports that bead SB may be suitable as a means forpre-carbon treatment of process solution.

Site K Column Series 1 had a similar overall effect as it did at site A(Table 13). There was significant removal of all metals by the firstcolumn which resulted in decreased binding of Ag and Cu and low amountsof Pb on the carbon. A majority of the Au and Ag was removed by thefirst column but it still resulted in an increase in total Au loading onthe carbon (over 10 times).

TABLE 13 Comparison of Column Series 1 and 2 at site K. Site K Au Ag CuPb Bead g/t % dist. g/t % dist. g/t % dist. g/t % dist. Carbon 0.02 —1.4 — 850 — 0.17 — Column DB50 5.5 91 10 77 7600 88 44 92 Series 1 SB0.28 5 2.5 19 660 8 3.5 7 Carbon 0.26 4 0.50 4 350 4 0.49 1 Column SB0.74 50 6.7 90 1400 62 5.8 78 Series 2 Carbon 0.74 50 0.76 10 856 38 1.622

For Column Series 2 there was a significant level of Ag, Cu and Pbremoved by the first column but it did not lead to decreased loading ofthese metals on the carbon. Though Ag levels did decrease, similar Cuand higher Pb amounts were observed on the carbon. However, there wasstill a significant increase in Au loading on the carbon (over 30 times)compared to carbon alone.

Although improved carbon Au loading was observed in Column Series 2 atboth sites, a discrepancy between the total amount of loaded Au wasestablished between Column Series 1 and 2. This emphasizes the need toperform further tests that do not limit the amount of process solutionthrough the columns. This would lead to conditions closer in line tothose on site, and in turn, a better understanding of how these seriesof columns will perform. Overall though, using beads in series withcarbon can significantly increase the carbon loading of Au and decreasethe loading of Cu and Pb. One of skill in the art could readily modifythe upstream bead columns to decrease the Au removal, to increase theamount of Au available to be bound by the carbon.

G. Column Series: Bead Optimization

As noted above, it was observed that bead DB120 was able to removesignificant amounts of Au from sites A and K. Similar to carbon, DB120also binds Cu in high amounts. Therefore, a bead was added prior tosolutions passing through the columns to remove Cu and potentiallyincrease Au selectivity. Based on the prior column results, it wasdecided to again test beads DB50 and SB, this time with DB120. For bothsites A and K, Column Series 3 (bead DB50 then SB upstream of beadDB120) and Column Series 4 (bead SB upstream of bead DB120) were testedto potentially replace carbon (Table 14 and Table 15).

TABLE 14 Comparison of Column Series 3 and 4 at site A. Site A Au Ag CuPb Bead g/t % dist. g/t % dist. g/t % dist. g/t % dist. DB120* 8.34 —4.44 — 3090 — 211 — Column DB50 15 79 4.8 53 7700 80 1100 98.9 Series 3SB 2.4 13 2.7 30 920 9.6 2.5 0.2 DB120 1.7 9 1.6 18 1000 10.4 9.1 0.8Column SB 2.3 39 3.3 57 870 37 77 68 Series 4 DB120 3.6 61 2.5 43 150063 37 32 *Calculated metal loading based on percent removal.

TABLE 15 Comparison of Column Series 3 and 4 at site K. Site K Au Ag CuPb Bead g/t % dist. g/t % dist. g/t % dist. g/t % dist. DB120* 0.72 —1.14 — 828 — n/a — Column DB50 3.5 80 9.4 70 6200 79 19 60 Series 3 SB0.56 13 2.9 21 660 8 1.9 6 DB120 0.34 8 1.2 9 950 12 11 34 Column SB0.82 56 4.2 72 1000 50 5.6 32 Series 4 DB120 0.65 44 1.6 28 990 50 12 68*Calculated metal loading based on percent removal.

When observing site A, Column Series 3 had a similar overall effect asColumn Series 1. There was significant removal of Cu and Pb by the firstand second columns, which decreased both metals dramatically on beadDB120. The upstream beads were responsible for significant Au loadingwhich resulted in relatively low Au loading on bead DB120. However,compared to carbon at this site with the same beads upstream, there wasover 10 times more Au captured on bead DB120 (FIG. 11). Similar trendswere observed when comparing Column Series 4 and Column Series 2 at thesame site. Again, bead DB120 captured more Au than carbon in the sameseries. Upstream, bead SB bound a high amount of Ag, Cu and Pb whichdecreased the amount of these metals on bead DB120, compared to DB120alone. Although a decrease is identified, DB120 still bound these metalsin a relatively high amount. One of skill in the art can readily improvethe selectivity of these beads.

Results of Column Series 3 at site K were similar in trend to thoseobserved at site A. Beads DB50 and SB removed large amounts of allmetals, however, at this site it did not result in lowered amounts ofmetals on bead DB120. In the end though, higher Au loading was observedon bead DB120 than carbon alone and slightly higher than carbon in thesame series. For Column Series 4, there was an increase in Au loading onbead DB120 compared to Column Series, but not to carbon in the sameseries. Throughout the series, bead DB50 consistently showed significantbinding of Cu (˜7 g/kg). The inventors predict that increase solutionvolumes will increase capacity for Cu binding by bead DB50.

These results show that bead DB120 could potentially be used as areplacement for a carbon-based process for capturing Au. Although theseColumn Series established that bead DB120 exhibited higher binding ofnon-target metals than carbon in the same series, it still possessed ahigher Au loading ability. Furthermore, extremely large differences wereobserved when comparing the loading capabilities of carbon and beadDB120 not in series (0.22 vs. 8.34, 0.02 vs. 0.72, g/t at sites A and K,respectively). This higher Au loading compared to carbon was alsoobserved with bead DB50 when it was used as the first column in ColumnSeries 1 and 3. Bead DB50 did not show a specific selectivity to Au asit also continuously loaded more Cu and Pb than carbon. One of skill inthe art could readily modify the column series to increase Au capacityand selectivity.

IV. Conclusions

Results obtained from testing at the mining operation were able to meetand exceed anticipated results based on prior laboratory testing. Thepresently disclosed innovative bead technology demonstrated asignificant ability to reduce non-target metal concentrations in processsolutions. This combined with the capability of the beads to alsocapture Au, has the potential for various possibilities. Numerous testswere performed to determine the performance, selectivity and loading ofthe beads.

Metal and CN concentrations were evaluated at various sites throughoutthe complex. As expected, metal concentrations varied throughout theprocess and a wide range of CN concentrations and pH levels wereobserved. From these results, it was determined that site K provided agood environment to test all beads to determine performance differences.

When testing all variation of beads, it was observed that smaller beads(3 mm) had higher removal percentages than larger beads (5 mm);supporting that increased surface area produced better results.Differences were detected when comparing heat treatment techniques forbead preparation as well as comparing beads prepared at varying pHlevels. Based on these results, select beads were further tested at twoadditional sites. Sites A and F possessed differing concentrations ofmetals and CN to each other and site K, and offered three sites withvarying conditions to further determine the differences in performanceof the beads. Similar trends in results were observed. Higher amounts ofAu removal occurred at site A. The increase was attributed to higherconcentrations of Au and lower concentrations of CN, allowing forincreased removal. Through these tests, it was determined that bead WBSexhibited the best overall performance and was tested at all sites todetermine if there were trends in performance or selectivity based onsite location.

When comparing all sites, bead WBS demonstrated a wide range of removalfor all metals. Predominantly, a higher removal of metal occurred whenCN concentrations were low and conversely, a lower removal whenconcentrations were high. While without wishing to be bound by theory,through testing of bead WBS at all sites, water chemistry and CNconcentration had just as significant an impact on performance as beadtype. Therefore, attempts were made to control the impact on CNconcentration and pH levels had on bead performance.

At sites A and K, it was demonstrated that by destroying the CN insolution removal percentages were improved, especially with Ag and Cu.All metals saw increased removal at both sites with CN destruction (withand/or without pH adjustment). These water optimization tests show thatplacement of beads is the most effective if located at sites where lowCN levels are present.

As an alternative to beads, unencapsulated Euglena was tested as asolution pretreatment technique to attempt to enhance the Au and Agloading capabilities of carbon. Contact of the solution with Euglenaprior to carbon demonstrated increased Au loading on the carbon. Thiswas similarly attempted with beads to try and eliminate non-targetmetals prior to carbon contact and ultimately improve Au adsorption.

In Column Series 1, beads DB50 and SB were used in series at sites A andK prior to carbon contact and removed significant amounts of Cu and Pb.This resulted in lowered amounts on the carbon. At site K carbon loadingincreased (0.02 vs. 0.26, g/t). Similar results were shown for ColumnSeries 2, when bead SB was used by itself prior to carbon contact. Auloading on the carbon improved significantly at both sites A (0.22 vs.2.8, g/t) and K (0.02 vs. 0.74, g/t) over carbon alone. Bead DB50demonstrated a higher than desired affinity for Au, affecting the amountavailable to be bound by carbon. However, it did consistently bindsignificant amounts of Cu (˜7 g/kg), which approaches industrystandards. One of skill in the art can readily decrease the affinity forAu while increase the affinity for non-target metals of upstream beads,which is predicted to significantly improve carbon binding abilities.

Example 2C: Batch Tests with Artificial Solutions

Batch tests were performed with artificial metal solutions to assessmetal removal capabilities of beads WBS and SB. 1.5% sodium alginate and1.5% calcium chloride solutions were used for encapsulation. For WBSbeads, a ˜4:1 mixture of 1.5% sodium alginate solution:wet biomassmixture was used. In addition, for SB beads, a ˜1:1 mixture of 1.5%sodium alginate solution:spent media solution was used. Single metal andmulti-metal solutions were tested. Results for single metal solutionsare the average of three tests and for multi-metal solutions, fourtests. Tests were run in flasks for 24 hours while being agitated onshaker tables. Solutions and solids were assayed by ICP-MS. Artificialmetal solutions were prepared as follows (Table 16A):

TABLE 16A Artificial metal solutions Testing For Solution Conc. (mg/L)pH Reagent Used Cooper (Cu) ~750 4.5 CuSO₄•5H₂O Lead (Pb) ~250 5.5Pb(CH₃CO₂)₂•3H₂O Gold (Au) ~5 2.6 Au stock (in 0.5M HCI) Silver (Ag) ~115.9 AgNO₃ All Cu: 573, Pb: 8.91, 4.8 As above, except AuCI₃ Au: 0.428,Ag0.984

In general, WBS and SB beads were, responsible for a similar percentremoval except when it came to single metal solutions of copper andgold, where WBS beads removed a slightly higher percentage (Table 16Band C). However, SB beads consistently loaded a larger amount of metalon a per dry bead mass basis.

TABLE 16B Results from single metal solutions. Bead Type Removal (%)Loading (g/t) Copper Removal WBS 23.1 41133 SB 13.6 110000 Lead RemovalWBS 91.5 48100 SB 92.6 596667 Gold Removal WBS 99.1 1009 SB 71.3 3329Silver Removal WBS 47.7 210 SB 45.2 317

TABLE 16C Results from multi-metal solution (Removal, %) Bead Type Cu PbAu Ag WBS 32.1 80.8 89.7 48.3 SB  31.8 83.8 87.4 49.0

Example 2D: Desorption, Reuse and Carbon Pretreatment Desorption

To help assess the capability of encapsulated Euglena and/or Euglenaexudates being used for commercial metal removal applications, metaldesorption tests were performed to determine the potential to removemetal bound to the beads; which would then allow the beads to be used toremove more metal. The beads were prepared as in Example 2C. Tests werefirst performed on beads after contact with artificial multi-metalsolutions with four different desorbents of the same molarconcentration: 0.2 M hydrochloric acid (HCl), 0.2 M sodium hydroxide(NaOH), 0.2 M nitric acid (HNO₃), and 0.2 M sulphuric acid (H₂SO₄).These desorbents, at two different concentrations (except for NaOH, onlyat 0.1 M), were then tested on beads after contact with mine solution.Two other desorbents, potassium cyanide (KCN) and thiourea, were testedfor selective removal of gold from beads. Successive rounds of testing,with desorption of metals in between, were then conducted to demonstratethat the ability to remove metals can be just as effective afterdesorption as using fresh beads. For these reuse tests, HNO₃ was used asthe desorbent between rounds of mine solution contact.

For desorption of metals after contact with multi-metal solution, thethree acids (HCl, HNO₃, H₂SO₄) were more effective overall than NaOH(Table 16D). However, NaOH was significantly better than the three acidsat desorption of Au. All three acids had similar success at desorptionof Cu and Pb from both WBS and SB beads, while HCl had an increasedability to desorb Ag from the beads. While for the most part both beadsreleased metals similarly, desorption of Ag from SB beads compared toWBS after contact with any of the acids was considerably increased. NaOHwas slightly better at desorbing all metals from bead WBS than bead SB.

TABLE 16D Desorption - multi-metal solution (desorption, %)Desorbent/Bead Type Cu Pb Au Ag 0.2M HCI/WBE 77.3 100 1.7 56.3 0.2MHCI/SB 78.0 96.1 13.5 100 0.2M NaOH/WBS 77.3 100 1.7 56.3 0.2M HCI/SB78.0 96.1 13.5 100 0.2M NaOH/WBS 25.0 74.3 100 50.0 0.2M NaOH/SB 14.367.6 70.4 39.0 0.2M HNO₃/WBS 77.2 93.1 9.0 11.9 0.2M HNO₃/SB 73.8 73.29.9 39.3 0.2M H₂SO₄/WBS 74.1 97.3 12.2 11.5 0.2M H₂SO4/SB 79.4 90.2 9.646.1

For desorption of metal from beads WBS and SB after contact with minesolution, an increase in desorbent concentration from 0.1 M to 0.25 Mwas able to desorb increased amounts of metals in almost all cases(Table 16E). Similar to the multi-metal solution, NaOH was lesseffective at desorbing metal compared to the acid desorbents, exceptwhen it came to Au. The three acids were more effective at desorbing Cuand Pb than Au and Ag. When comparing the two beads, all desorbents weremore successful at desorbing metals from bead SB than WBS. Overall, 0.25M HNO₃ shows to be the preferred desorbent for bead SB and 0.25 M HCland 0.25 M HNO₃ are the preferred desorbent for bead WBS. These resultsshow metal desorbed from encapsulated Euglena and Euglena exudates afterthey have removed metals from effluent solutions.

TABLE 16E Desorption - mine solution (desorption, %) Desorbent/Bead TypeCu Pb Au Ag HCI 0.1M/WBS 7.3 77.2 3.3  3.4 0.25M/WBS 81.9 97.0 18.3 n/a0.1M/SB 38.9 92.0 48.2 64.0 0.25M/SB 100 100 46.7 n/a NaOH 0.1M/WBS 20.28.8 23.7  2.1 0.1M/SB 26.0 4.3 26.0 12.9 HNO₃ 0.1M/WBS 21.3 67.8 9.0 2.3 0.25M/WBS 93.4 59.2 30.4 n/a 0.1M/SB 17.3 90.5 8.8 19.1 0.25M/SB100 100 99.4 n/a H₂SO₄ 0.1M/WBS 16.7 69.8 11.6  1.6 0.25M/WBS 88.3 54.621.5 n/a 0.1M/SB 62.6 47.8 46.3 65.4 0.25M/SB 100 83.0 65.1 n/a

Selective Au Desorption

As shown in Table 16F, at contact time of 18 h, increasing theconcentration of KCN results in increased desorption of Au from thebead. Other metals were also desorbed, but desorption of Cu and Pbdecreased slightly as the concentration of KCN was increased. Agdesorption remained essentially the same as the concentration of KCNchanged. Increasing the concentration of thiourea also increased thedesorption of Au. A shorter contact time with the same concentration ofthiourea was less effective at desorption of Au, while also desorbingmore Cu and Ag.

TABLE 16F Selective Au desorption - mine solution with WBS beads(desorption, %) Desorbent Cu Pb Au Ag 0.0077M KCN 63.7 30.5 32.9 51.40.1M KCN n/a 31.8 52.7 56.3 0.2M KCN (1 h) 42.3 18.5 66.7 43.6 0.3M KCN(1 h) n/a 17.8 70.9 51.9 0.1M Thiourea + 0.1M HCI 32.3 83.2 20.9 47.70.1M Thiourea (1 h) 100 39.8 54.0 12.5 0.2M Thiourea (1 h) 100 2.7 71.728.0 0.2M Thiourea 72.7 4.4 84.8 14.4

Reuse

The metal removal ability of WBS beads was not affected after desorptionof metals (Table 16G). This demonstrates that a desorbent, such as NHO₃,can effectively remove bound metals and free up binding locations on thebeads for subsequent metal removal without affecting the beads efficacyfor at least 3 consecutive contacts with solution.

TABLE 16G Reuse - mine solution (% removal; average of three tests) WBSbeads Cu Pb Au Ag 1^(st) round 36.6 n/a 7.2 78.4 2^(nd) round 45.0 50.07.8 83.2 3^(rd) round 34.6 66.7 2.6 81.0

Pretreatment Carbon Tests

Tests were performed to determine the benefit of using beads WBS and SBas a solution pretreatment prior to contact with carbon, for example,with train solution that is taken just before mining process waterentering carbon trains. Without wishing to be bound by theory, beadswould remove the non-target metals (Cu and Pb), decreasing the loadingof these metals on the carbon which would result in higher loading oftarget metals (Ag, and especially Au) on the carbon. Tests were runconcurrently with no bead pretreatment and carbon contact alone in orderto assess the benefit of bead pretreatment. Bead contact was for 6hours, while carbon contact was for 16 hours. Results are shown as gramper tonne loading of metals on the carbon by solution type and beadpretreatment (Table 16H).

TABLE 16H Carbon Tests - Loading (g/t). Au Ag Cu Pb Train Solution BeadWBS pretreatment 56 31 8400 104 Bead SB pretreatment 53 40 8200 62 Nobead pretreatment 56 43 8150 420 Pond Solution Bead WBS pretreatment 5193 9400 1 Bead SB pretreatment 51 113 9400 0 No bead pretreatment 47 12912000 3

Pretreatment with either bead results in less Pb from train solution(similar to site A solution) being loaded onto carbon, demonstratingthat they have the properties to be used in a filter-like capacity toeliminate non-precious metal binding on the carbon without affecting Auloading. This is especially the case with bead SB, which when used as apretreatment significantly decreases the amount of Pb loaded onto thecarbon without significantly affecting the Au and Ag loading. Contactingtrain solution with WBS or SB beads prior to carbon contact had noeffect on carbon loading of Au or Cu. As for pretreatment of pondsolution (similar to site K solution) with bead WBS and SB, thisresulted in a decrease in binding of Cu and Pb onto carbon whileslightly increasing gold loading. This shows that the encapsulatedEuglena or Euglena exudates possess the properties to function as apretreatment filter to eliminate non-target metals to improve the Aupurity on carbon.

Collectively these results demonstrated that the disclosed beadtechnology are useful. In particular, the methods, uses and biosorbentsdisclosed herein are useful for remediation or recovery of metals frommining process water such as from carbon trains, resin columns, pondssuch as excess, intermediate, pregnant and tailing ponds, leach pads,electrowinning cells, tanks such as flotation, Merrill Crowe, solidphase extraction and pregnant tanks, as well as mining process waterleaving or entering carbon trains, resin columns, ponds such as excess,intermediate, pregnant and tailing ponds, leach pads, electrowinningcells, tanks such as flotation, Merrill Crowe, solid phase extractionand pregnant tanks. The ability to capture both target and non-targetmetals is significant. These beads possess the ability to be analternative to the current technologies available for metal recoveriesand are believed to be more efficient, more flexible and morecost-effective to work complementary to current techniques, such ascarbon columns or even replace them. One of the skill in the art canreadily modify the disclosed bead technology to increase metalselectivity and loading capacities.

Example 3: Structural and Molecular Characterization of LyophilizedEuglena gracilis Cells I. Introduction

As shown in Examples 1 and 2, the algal flagellate Euglena gracilis isan efficient biosorbent that warrants further investigation on astructural and molecular level. E. gracilis can grow in the presence andabsence of light, and also in aerobic and anaerobic conditions. E.gracilis can grow in a broad range of pH values 2.5-8, and is naturallyfound in freshwater, seawater and brackish waters. E. gracilis has thepotential to be a viable biosorbent for the biosorption of metals,metalloids and REE.

In the present disclosure, molecular structural analysis of freeze-dried(i.e. lyophilized) E. gracilis has been conducted by electrosprayionization high-resolution Fourier transform orbitrap mass spectrometry(Q-Exactive ESI Orbitrap). The present study discloses structurally andon a molecular level the presence of key compound classes and functionalgroups within E. gracilis, using a combination of spectroscopic (FTIR)and mass spectrometry techniques (HR-MS) and the growth phase andconditions that are best for the enhancement of compound classes forbiosorption potentiality.

II. Experimental Procedures A. Algal Cultures

Euglena gracilis Z, was axenically cultured at 29° C. under a 12 h light(54,000 lx) and 12 h dark cycle, and also cultured in 24 h darkness at29° C. in a dark chamber (ThermoScientific). E. gracilis was grown inEuglena gracilis medium (or EGM; Winters et al., 2016) with and withoutglucose supplementation (20 g·L⁻¹) in 1 L Erlenmeyer flasks, with 500 mLof culture medium. E. gracilis was collected in the exponential andstationary phases of growth. Cells were centrifuged at 4000 rpm for 6min and washed thoroughly with Milli-Q water to remove any culturemedium from the cells. The lyophilized cells were homogenized usingmarble mortar and pestle prior to structural and molecularcharacterization.

B. FTIR Analysis

FTIR spectra of lyophilized E. gracilis were obtained by using aThermoNicolet 380 ATR FTIR spectrophotometer equipped with the EZ OmnicSpectra software (ThermoScientific). The FTIR spectrum was recorded overa range of 4000 cm⁻¹ to 400 cm⁻¹. Thirty-two scans were acquired at aresolution of 4.000, aperture of 100.00, and mirror velocity of 0.6329.

C. AF4 Analysis

The AF4 system was a Postnova Analytics AF2000 Focus (Salt Lake City,Utah, USA) equipped with an online Shimadzu UV-Vis detector (SPD-M30A)operated at 254 nm. A 1 kilodalton (kDa) regenerated cellulose and a 350μm spacer were used. The eluent (10.1 mM NaCl) was prepared daily. Ablank sample of carrier fluid was run before each sample to ensure theabsence of memory effects. 200 μl samples were injected via a manualinjector valve. Fractions A-D (FIG. 17) were collected at the retentiontimes of 7.5-8, 8-9, 9-10, 10-11 min, respectively, whereas the combinedfraction was collected between 7.5-11 min. The unfractionatedsupernatant was also compared with the combined fraction. The fractionscollected, and unfractionated samples were refrigerated at 4° C. andanalyzed within 4 days. The replicates of AF4 fractionation were donewithin 2 days (1 replicate per day).

D. Q-Exactive Orbitrap Analysis

All high-resolution mass spectrometry analyzes were conducted on aQ-Exactive Orbitrap (ThermoFisher) housed at the Trent Water QualityCentre (Peterborough, Ontario, Canada). The lyophilized E. gracilis (1mg·L⁻¹) samples were diluted with Milli-Q water and LC-MS grade methanolto give a final concentration of 50:50 methanol:water. The acquisitionparameters in positive ion mode were similar to that of Mangal et al.(2016) except that the flowrate was set at 25 μl/min. All samples werespiked with sodium trifluoroacetate (NaTFA) as a calibration standardwith a lock mass of 158.96403 in positive ion electrospray mode. Thescan range was from 150 to 2000 m/z.

The van Krevelen analysis was completed using an in house Matlabscripts. The different compound classes were defined as lipids(0.01≤O/C≤0.1; 1.5≤H/C≤2.0), unsaturated hydrocarbons (0.01≤O/C≤0.1;0.75≤H/C≤1.5), condensed aromatic structures (0.01≤O/C≤0.65;0.25≤H/C≤0.75), protein (0.1≤O/C≤0.65; 1.5≤H/C≤2.3; N≥1), lignin(0.1≤O/C≤0.65; 0.75≤H/C≤1.5), tannins (0.65≤O/C≤0.85; 0.75≤H/C≤1.5), andcarbohydrates (0.65≤O/C≤1.0; 1.5≤H/C≤2.5) (Ohno and Ohno, 2013; Mangalet al., 2016). Lignin and protein compound classes were comparable inpositive ion electrospray mode rather than negative ion electrospraymode, which is advantageous to this study.

III. Results and Discussion A. Growth Curves

The growth rates varied in both exponential and stationary phases underdifferent growth and media conditions (Table 17). Light grown cells withthe supplementation of glucose surpassed the other media conditions ingrowth rate, at both exponential and stationary phases of growth. Thegrowth rates seen in light grown cells with glucose supplementation wereas expected, as glucose would be metabolized quicker than the other EGMconstituents.

TABLE 17 Varying Euglena gracilis growth rates in both exponential andstationary phases under different growth and media conditions. Growthconditions Day Light [μ] Light (Glucose) [μ] Dark [μ] Dark (Glucose) [μ]0 2 0.32 0.90 −0.27 0.24 4 0.47 0.78 0.25 0.35 6 0.35 0.62 0.24 0.30 80.30 0.56 0.15 0.28 10 0.18 0.47 0.18 0.20 14 0.17 0.30 0.08 0.11

B. Functional Groups

FTIR spectra of the Euglena cells showed five distinct absorption bandswith the wavenumbers ranging from ˜950 to 1700 cm⁻¹ (FIG. 12). Thesebands were assigned to specific functional groups (carboxylic, phenolic,and amide groups). The carboxylic group (—COOH) was characterized by theC═O stretching at 1710±15 cm⁻¹ and with C—O stretching in the 1265±55cm⁻¹ region. Amide 1 (C═O stretching) was found in Euglena cells in the1650 cm⁻¹ region. Amide II and III were found in the ˜1550 and 1260±40cm⁻¹ regions, respectively. Lignin denoted by the phenolic groups withasymmetrical stretching of the C—O bond was also found at 1230 cm⁻¹.Similar peaks were found in both light and dark conditions, with andwithout glucose supplementation (FIG. 12). While not wishing to belimited by theory, this finding provides that regardless of the growthconditions and phases, similar functional groups were present within thecells grown in dark and light conditions.

C. Structural Analysis Influence of Photoperiod Duration (Light Vs DarkGrown Cells)

The average abundances of CHO and CHON compounds in exponentially grownlight grown cells EGM medium were 9.8 and 28.1% (FIG. 13), respectively,which was comparable to that found in E. gracilis dissolved exudates (14and 21% respectively; Mangal et al., 2016). The abundance of sulfurcontaining compounds varied significantly between cellular and exudatemetabolites. The CHONS species were 48% more abundant in cells than inexudates (Mangal et al., 2016). Higher percentages of the CHO molecularspecies were seen in dark grown cells (24.4-30.9%) than in light growncells (9.8-12.2%), regardless of growth phase and media conditions (FIG.13). The light grown cells were enriched in CHOS and CHON. Togetherthese results showed heteroatom-rich metabolites were preferentiallyfound in light grown cells. It is likely that organelles that are highlyspecialized to function in light conditions, such as paramylon andchloroplast, would not be active or absent in dark conditions.

The CHON₁₋₂ molecular species were more prominent in light grown samples(25.1-28.5%) than dark grown samples (14.0-17.2%) in all mediaconditions (FIG. 13). The CHOS₁₋₂ molecular species were present in allsamples with dark grown cells possessing more of these species(11.8-15.5% vs 13.3-18.9% in light and dark grown samples, respectively;FIG. 13). The CHON₁₋₂S₁₋₂ molecular species were present in all thesamples analyzed (40.3-48.8 vs 37.9-47.7% in light and dark grownsamples, respectively; FIG. 13). Light grown cells showed lower H/C andhigher double bonded structures (DBE) values than dark grown cells(p<0.05; Table 18), showing the presence of more aromatic functionalgroups in light grown cells. Dark grown cells showed higher O/C and S/Cratios (p<0.05), indicating that these cells were more oxygenated andsulfur-rich. No significant difference in aromaticity index (AI) wasfound between dark grown and light grown cells (p>0.05). This markeddifference provides, while not wishing to be limited by theory, thatlight promotes the formation of more CHON₁₋₂ molecular species,regardless of media conditions or growth phase.

TABLE 18 Elemental ratios and abundances, AI and DBE of cells grown indifferent culture conditions (light vs dark, normal vs supplementedmedium). Positive Ion Mode O/C H/C N/C S/C AI DBE Error (ppm) % C % H %O % N % S Light Conditions Exponential (Normal Media) 0.206 1.417 0.0450.023 0.183 12.854 0.358 59.662 7.465 2.339 25.100 5.457 Exponential(Supplemented Media) 0.212 1.375 0.045 0.024 0.191 11.516 0.343 58.4696.891 1.831 27.675 4.959 Stationary (Normal Media) 0.225 1.401 0.0480.032 0.077 10.948 0.139 60.053 7.295 2.181 25.792 4.700 Stationary(Supplemented Media) 0.199 1.394 0.045 0.027 0.126 11.201 0.296 61.3767.136 1.856 24.772 4.883 Dark Conditions Exponential (Normal Media)0.269 1.544 0.038 0.066 0.071 6.900 1.009 64.645 8.390 3.591 18.4134.974 Exponential (Supplemented Media) 0.246 1.490 0.037 0.068 0.1317.518 0.947 63.687 8.251 3.274 20.101 4.707 Stationary (Normal Media)0.248 1.489 0.037 0.069 0.131 7.607 1.030 64.671 8.648 3.178 18.8154.707 Stationary (Supplemented Media) 0.245 1.480 0.036 0.067 0.1397.751 1.011 64.975 8.714 3.274 18.399 4.657

A marked difference in compound class abundances within dark grown andlight grown samples was found (FIG. 14). Protein and lignin compoundclasses were predominant in E. gracilis cells. The protein compoundclass accounted for 39 to 50% of the total assigned peaks in E. graciliscells. Lignin was more abundant in dark grown cells than in light growncells. Without wishing to be bound by theory, the 1.43-fold differencein lignin abundance was likely due to the photobleaching of ligninstructures in the light grown cultures (Opsahl and Benner, 1998; Helmset al., 2008; Herres et al., 2009; Spencer et al., 2009). Dark growncells had a higher overall percentage of lignin, carbohydrates, andaromatics. Lower percentages of lignin and aromatics in light growncells are likely due to the phototransformation of lignin and aromaticcompounds (Lu et al., 2016; Medeiros et al., 2015). Higher carbohydratecompound class in dark grown conditions was likely due to more paramylonformation (Matsuda et al., 2011). Lipid compound classes percentages indark grown and light grown cells of E. gracilis (4.8-16.4%, and12.8-15.3%, respectively) were similar.

Influence of Growth Phase

The composition of cellular metabolites was growth phase dependent(Table 18; FIG. 14). Principal component analysis (PCA) based onelemental ratios and AI (FIG. 15) showed that exponentially grown darkgrown cells in normal media influenced by O/C ratio, indicating thatthese cells were more oxygenated. Dark grown conditions produced moreoxygenated and sulfonated groups (higher O/C and S/C ratios), whilelight grown conditions favored aromatic and unsaturated structures (FIG.15). The dark grown cells in exponential phase contained 20% moreproteins than the light grown cells. This contrasted with the stationaryphase where more proteins were associated with light grown cells thanwith the dark grown cells (46.6-47.7% vs 39-42.9%, respectively; FIG.15). Without wishing to be bound by theory, this could be due to thedifferentiation of proplastids to photosynthetic plastids when exposedto light (Schwartzbach and Shigeoka, 2017). Cell lysis is oneexplanation for the presence of more proteins and higher % N in thestationary phase (Table 18). Protein percentages was highest in darkgrown Euglena cells with glucose at the exponential phase (50%),congruent with higher % N in supplemented medium (Table 18). Dark growncells were less enriched in protein at the stationary phase whereas thereverse was seen for light grown cells. Without wishing to be bound bytheory, Fogg (1957) stated that in the exponential phase, light grownalgae had a high photosynthetic rate and the products of photosynthesiswere used mainly for the protein synthesis. By the end of theexponential phase, the photosynthetic rate would significantly bereduced, and photosynthetic products would become increasingly divertedalong pathways other than that of protein synthesis to form “reserves”of lipid or carbohydrate (Fogg 1957). This process was congruent withthe reduction in protein abundances in the stationary phase compared tothe exponential phase for light grown cells. Differentiation ofproplastids into functional photosynthetic apparatus may have a role toplay in this as well.

When comparing percentage abundances of compound classes of Euglenaexudates, the cells showed similar abundance of carbohydrate, proteinand lignin but much higher abundances of lipids, condensed aromaticstructures and unsaturated hydrocarbons. Without wishing to be bound bytheory, higher abundances of carbohydrates, protein and lignin moleculesin cells were likely due to their role in cellular processes and/or asnutrient sources (i.e. lipid as storage, or for membrane function;proteins for structural integrity and involved in enzymatic reactions;carbohydrates for paramylon storage in E. gracilis) (Matsuda et al.,2011; Šantek et al., 2012).

Principal component analysis revealed significant differences incompound class composition of cells grown in different conditions (FIG.16). First and second principal components (PC1 and PC2) represented 59and 30% of the total variance explained. Positive PC1 indicatedsaturated compounds (lignin, aromatics) whereas negative PC2 showedproteinaceous material. The light grown and dark grown samples wereclustered separately with the dark and light grown samples displayingpositive and negative PC1 values, respectively. The light grown cellswere more aromatic in character, congruent with lower H/C and lower % H(p<0.05; Table 18). The increase in PC2 from the exponential tostationary phase in the dark grown cells indicated a decrease in proteinmaterial and an increase in lipid compounds. This contrasts with thelight grown cells where an enrichment in proteinaceous material wasfound in stationary phase relative to the exponential phase.Physiological modes could affect differences seen. This drastic changeis likely due to the difference in energy storage. The autotrophicorganisms can store energy as lipid whereas the dark grown organismsfavored protein storage (Schwartzbach and Shigeoka, 2017). Togetherthese results highlighted that growth conditions influenced structuralcomposition of cellular organic compounds.

D. Comparison of Unique Compound Classes and Molecular Species withLight and Dark Grown Cells

TABLE 19 Compound classes of unique (m/z) peaks Compound Classes ofunique (m/z) peaks in Percentages Unsaturated Condensed Lignin ProteinLipid Hydrocarbons Carbohydrates Tannins Aromatics Light Grown Cells(Unique Peaks) Exponential Phase vs Stationary Phase (in normal media)23.26 40.97 15.63 16.32 2.43 0.69 0.69 Exponential Phase vs StationaryPhase (both with Glucose) 29.00 41.00 9.50 13.00 6.00 1.00 0.50Exponential Phase vs Exponents Phase (Glucose) 22.69 42.02 15.13 15.413.08 1.12 0.56 Stationary Phase vs Stationary Phase (Glucose) 23.7545.00 12.50 12.92 4.58 1.25 0.00 Dark Grown Cells (Unique Peaks)Exponential Phase vs Stationary Phase (in normal media) 23.33 46.6720.00 6.67 3.33 0.00 0.00 Exponential Phase vs Stationery Phase (bothwith Glucose) 42.86 42.86 0.00 0.00 7.14 0.00 7.14 Exponential Phase vsExponential Phase (Glucose) 27.50 37.50 17.50 7.50 5.00 2.50 2.50Stationary Phase vs Stationary Phase (Glucose) 30.77 30.77 15.38 3.8511.54 3.85 3.85

The compound classes unique to all compared growth phases and mediaconditions were dominated by the lignin and protein compound classes,comprise of the majority of the unique peaks, which were notsignificantly different, except in dark grown samples when theexponential and stationary phases (with glucose) were compared forlignin (Table 19). Light grown samples have higher percentages of uniquepeaks for the unsaturated hydrocarbons compound class than dark grownsamples, in all compared media conditions. Dark grown samples showedhigher percentages of unique peaks of carbohydrate than light grownsamples when the exponential and stationary phases inglucose-supplemented media were compared. Unique condensed aromaticcompounds still were more prominent in dark than light grown cultures inall media conditions. Tannins were prominent in dark grown samples thanlight grown samples, but only when these conditions were compared:exponential phase versus exponential phase with glucose, and stationaryphase versus stationary phase with glucose.

E. AF4 Separations of Cellular Fractions

The AF4 fractograms were relatively consistent between replicatecultures (FIG. 17), suggesting comparable size distribution withingrowth condition. Significant differences in the AF4 fractograms werefound between light and dark grown cells, indicating differences inmetabolomics processes. The light grown cells have a more sophisticatedmachinery due to differentiation (Schwartzbach and Shigeoka, 2017).Light grown cellular fractograms (FIG. 17; top graph) showed acharacteristic peak at 7.8 min (fraction A) followed by a broad peak at9.3 min (fraction C). On the other hand, the dark grown cellularfractograms (FIG. 17; bottom graph) showed a sharp peak at 8.3 min(fraction B) and a very broad peak at 9.5-10.5 min (fraction C). Thehigher retention times suggested higher molecular weight in metabolitesisolated from dark cells, congruent with higher weighted average m/z indark cells.

Comparable number of m/z assigned features were found in unfractionatedand AF4 combined fractions (i.e. fraction A+ fraction B+ fraction C+fraction D) in dark cells whereas a reduction of 50% of the total m/zfeatures was found in AF4 combined light cell samples compared to theunfractionated samples (FIG. 18). Unfractionated and AF4 combinedsamples shared 187 and 339 common features in light and dark samples,respectively, representing 14-28% and 32-35% of the total assignedfeatures. The composition of light grown cells was more significantlyaltered by AF4 fractionation than the dark grown cells (FIG. 19). Forexample, the lipid abundance increased from 26 to 35% after AF4fractionation of light grown cells whereas it remained unchanged in darkgrown cells. Although its abundance decreased slightly after AF4fractionation, the number of lignin formula remained relatively constantin dark grown cells. AF4 procedure includes a focusing step in which theanalytes of interest (i.e. cellular material) are concentrated on theAF4 membrane whereas the solvent and smaller analytes are eliminated.Without wishing to be bound by theory, causes of this includes ligninformula preferentially associating with higher m/z peaks in dark growncells which were less likely to be lost during focusing through the 1kDa AF4 membrane. The mean m/z average of protein formulae increasedafter AF4 fractionation (473 to 918 m/z), a result of a preferentialloss of low molecular weight compounds during fractionation.Approximately 40% and 71% of protein formulae were lost in dark andlight conditions, respectively.

Although most m/z values were found at 100-1000 m/z (77.4-92.1% of totalassigned formulae), the abundance of features in the combined fractionof light cells decreased in the 100-700 m/z range but increased athigher m/z (800-1400 m/z) compared to the unfractionated light cells(FIG. 20). This contrasts with the dark conditions where the m/zdistribution did not change drastically after AF4 fractionation. Theloss of lower m/z metabolites in light conditions were likely notretained on the 1 kDa membrane during the AF4 focusing step.

934 to 2648 features (shown as “assigned peaks” in Table 20) weredetected in cell fractions ranging from 150 to 1500 m/z. 349-767features (27.5-72.7%) were commonly found in the biological duplicates.The replicates of the AF4 fractions shared 27-73% (327-715 features) and46-56% (501-701 features) of the features associated with light and darkconditions, respectively. Comparable common features were found in amicrobial study (Becker et al., 2014). The greater variability in sharedfeatures was found in the light cells fractions, likely due touncontrollable differences in growth conditions and/or small variationsin growth stage at the time of processing heterogeneity (Becker et al,2014). The genetic machinery catered for light cells is suggested to bemore sophisticated (O'Neill et al., 2015) and thus more heterogeneous inlight than dark.

TABLE 20 Abundances of unique and common m/z peaks (i.e. features), andnumber of assigned peaks in light and dark grown biological replicates.Unique Unique Common features features features of of of AssignedReplicate 1 Replicate 2 Replicates Peaks Light Fractions A 28.7 39.548.7 1054 B 72.8 33.5 23.9 1366 C 50.9 16.9 44.7 1601 D 14.9 16.7 72.7934 Combined 59.0 47.7 29.9 1169 Unfractionated 70.4 59.5 20.6 2648 DarkFractions A 31.3 28.7 53.8 1037 B 13.3 38.0 56.6 1238 C 35.1 19.2 56.21153 D 21.5 38.9 52.4 1033 Combined 19.7 48.2 46.0 1090 Unfractionated27.4 28.0 56.6 1355

Venn diagram analyses of the four AF4 fractions revealed only 79 (11.7%)and 48 (6.3%) common features in all light and dark fractions,respectively (FIG. 21). Most features were unique to one specific AF4fraction, showing that the AF4 method allows the isolation of uniquecellular features. The light fractions showed more unique features thanthe dark fractions (except fraction B), with light fraction D showingthe most unique features (312).

In terms of compositional abundance, protein, lipid, lignin, unsaturatedhydrocarbons, and carbohydrates dominated the composition of uniquepeaks in fractions grown under light and dark conditions (FIG. 22). Thelower lipid abundances in dark grown cells relative to light grown cells(p<0.05; except fraction B), without wishing to be bound by theory,could be explained by the diversion of lipids to photosynthetic pathwaysupon differentiation of the cells (Matsuda et al., 2011; Schwartzbachand Shigeoka, 2017). Comparable results were found in previous studies(Rosenberg and Pecker, 1964; Constantopoulos and Bloch, 1967) wherelipid content in Euglena was altered with increasing light. Lipidcharacteristics such as higher reduction state, hydrophobic characterand the ability to be efficiently packed into the cell and generate highamounts of energy upon oxidation, suggest this class is a reserve forrebuilding the cell after stress has been alleviated (Moralez-Sanchez etal., 2016). Without wishing to be bound by theory, this could explainwhy lipid abundances were higher in Euglena dark fractions than lightfractions as previously shown (Li et al., 2014). The greater proteinabundance in light cells may be related to the active photosyntheticlight system as synthesis of the precursor molecules for the lightharvesting chlorophyll a/b binding protein of photosystem II (LHCPII)increased 50-100-fold upon exposure of dark grown resting Euglena cellsto light (Kishore and Schwartzbach, 1992).

When comparing AF4 fractions, the abundance of protein compounds inlight conditions decreased from fraction A to D (i.e. with increasingretention time and thus molecular weight; Schimpf et al., 2000), whilethe converse was found for lipid compounds (p<0.05; FIG. 22A). Theincrease in proportion of lipids in different cellular fractions (A-D)indicated larger size cellular material was lipid-rich. A decrease inproportion of protein compounds in different fractions (A-D) could bedue to organelles of higher molecular weight exhibiting lower proteincontent. No significant change in protein and lipids was found in AF4dark fractions (p<0.05; FIG. 22B). The dark and light cellular fractions(A-D) did not show any significant differences in double bondedstructures (DBE) or O/C ratios, indicating comparable levels in aromaticand oxygenated compounds. Significant differences in m/z values werefound (p<0.05), with higher m/z values in the dark fractions. ThisExample shows that fraction C from dark grown cells was the mostenriched in lignin+protein+carbohydrates (p<0.05), which are compoundclasses associated with metal binding.

PCA analysis of elemental ratios (O/C, H/C, N/C, and S/C), AI_(mod),double bonded structures (DBE) and m/z in fractions A-D revealeddistinct patterns between light and dark fractions (FIG. 23). The firsttwo principal components (PC) represented 52.4% and 23.4% of the totalvariance, respectively. Light and dark fractions were mainly separatedalong the PC2 axis. The dark fractions (except fraction A) werecharacterized by higher m/z and lower AI_(mod) than the light fractions,showing the significant differences in molecular composition of cellsgrown in light and dark conditions. The positive PC1 and PC2 scores oflight fractions B and C indicated the prevalence of O-, N- and S-richcompounds compared to the dark fractions B and C. Light fractions A andD were strongly shifted to the left quadrant, revealing their greaterabundance of DBE compared to light fractions B and C.

IV. Conclusions

The presence of carboxylic and proteins, functional groups typicallyinvolved in transition metal and rare earth metal (REE) binding, wereconfirmed by FTIR spectroscopy in exponential and stationary growthphases of dark and light grown cultures. No major differences inFTIR-based structural composition were found between growth phases. Thestructural composition of E. gracilis cells based on Orbitrap Q-Exactiveshowed that lignin, carbohydrate and aromatic were the most abundant indark grown conditions with glucose supplementation. This confirms thedifferences in physiological states of the cell, in different growth andculture conditions. Based on the results presented, conditions thatwould potentially facilitate the most efficient metal removal, inparticular for REE, would be dark grown E. gracilis cells grown withglucose supplementation at the exponential phase of growth. Usingconstraints to identify compound classes (lipid, protein, tannin,carbohydrate, aromatics, and unsaturated hydrocarbons) is a novel way ofdetermining the different families of compounds in algal cells. Thelignin, aromatics, protein and carbohydrate compound classes arepotential ones for REE removal via biosorption.

Molecular composition was different between AF4 fractions with thearomatic character being enhanced in some AF4 fractions. Based on thecomposition of fractionationed cells, fraction C of dark grown cells wasthe most favorable fraction for metal binding and thus metal removal andextraction.

A greater variability in features shared by AF4 fraction in cells grownin light conditions was found. Dark conditions were superior in terms ofthe cellular abundance of aromatic+protein+carbohydrates compoundscompared to light conditions, showing that Euglena cultured under thiscondition would be most useful for extraction of metals.

Example 4: Use of Diffusive Gradient in Thin Films (DGTs) Technologies,Dialysis Algal Cells or Exudates, for Metal Remediation in Waters I.Introduction

The size of the Euglena exudates may be small enough to diffuse throughE. coli cell membranes. E. coli cells have been used as biosensor of Hgmobilization (Chiasson-Gould et al., 2014). FIG. 24 shows thatbioluminescence induced by free 250 pM Hg or 250 pM Hg exposed toEuglena exudates in E. coli biosensor was comparable. Therefore, if theEuglena exudates are to be used in a filtration apparatus to removemetal, a system should be in place to ensure that the exudates do notdiffuse out of the apparatus. With this in mind, the use of dialysis bagwas investigated to determine whether it could prevent exudatesdiffusion. In the present disclosure, dialysis bag studies showed thatit was feasible to retain the exudates in the dialysis bags.

The size of the Euglena exudates may be small enough to diffuse throughE. coli cell membranes. E. coli cells have been used as biosensor of Hgmobilization (Chiasson-Gould et al., 2014). FIG. 24 shows thatbioluminescence induced by free 250 pM Hg or 250 pM Hg exposed toEuglena exudates in E. coli biosensor was comparable. Therefore, if theEuglena exudates are to be used in a filtration apparatus to removemetal, a system should be in place to ensure that the exudates do notdiffuse out of the apparatus. With this in mind, the use of dialysis bagwas investigated to determine whether it could prevent exudatesdiffusion. In the present disclosure, dialysis bag studies showed thatit was feasible to retain the exudates in the dialysis bags.

II. Experimental Procedures A. Algal Culture and Sample Preparation

Euglena gracilis cells were cultured at 29° C. under a 18:6 hrlight:dark (2000-2500 lux) cycle in 500 mL autoclaved modified Hutnermedium (Euglena gracilis medium or EGM; Dunstaffnage Marine LaboratoryCulture Collection of Algae and Protozoa). The cells (10⁶ cells/mL) wereharvested in the exponential growth phase and their exudates isolatedusing precombusted 0.2 μm nitrocellulose filters. Suwannee River fulvicacid (SRFA) standard obtained from the International Humic SubstancesSociety (IHSS) was diluted in Milli-Q water to a final concentration of7 mg·L⁻¹.

To determine influence of growth conditions on metal sorption Euglenagracilis cells were cultured as above in normal or Hg-amended (0.5 ppb)autoclaved EGM at pH 3.60. For cells cultured in Hg-amended medium (i.e.Hg-adapted cells), the medium was spiked with 0.5 ppb Hg in freshlyautoclaved medium at the end of each exponential phase. Normal andHg-adapted cells after 2-4 (younger generation) and 11 cycles (oldergeneration) were put in dialysis bags and immersed in normal autoclavedmedium spiked with metals and continuously stirred for three days. Theconcentrations of metal in the cells were measured using triplequadrupole ICP-MS (Agilent 8800, Trent Water Quality Centre).

B. Dialysis Bag

Five milliliter membrane tubing (0.1-0.5 kDa Biotech Cellulose Ester;Spectrum Labs) were closed using locking nylon membrane clamps. Beforeuse, the dialysis membranes were left in Milli-Q water overnight. Toassess the actual cutoff of the dialysis bags, a series of knownmolecular weight macromolecules (rhodamine-B, vitamin B-12, cytochrome,lysozyme and albumin) was used. Five milliliter of each macromoleculesolution (˜1 g·L⁻¹; n=2-4) was loaded in the dialysis bag andcontinuously stirred at 600 rpm for 3 to 5 days. Absorbances in themacromolecule feed solution (a_(initial)) and in the dialysate after 3to 5 days (a_(dialysate)) were measured on a 2550 UV-visible diode arrayspectrophotometer (Shimadzu) equipped with a 10-cm quartz cell. Therejection rate was calculated as follows:

${\%\mspace{14mu} R} = {\frac{a_{i{nitial}} - a_{dialysate}}{a_{i{nitial}}} \times 100}$

Five milliliter of samples (i.e. Euglena cells or exudates or SRFA) wereloaded into dialysis bags with nominal cutoffs of 0.1-0.5 kDa. The bagswere placed in a continuously stirred 500 mL beaker containing freshlyautoclaved (cells and exudates) or Milli-Q water (SRFA). The beakerswere continuously stirred for 3-5 days at room temperature.

C. Hg-Amended Growth Condition

The influence of growth conditions on metal sorption Euglena graciliscells was determined by culturing the cells as above in normal orHg-amended (0.5 ppb) autoclaved EGM at pH 3.60. For cells cultured inHg-amended medium, the medium was spiked with 0.5 ppb Hg in freshlyautoclaved medium at the end of each exponential phase. Normal andHg-adapted cells after 2-4 (younger generation) and 11 cycles (oldergeneration) were put in dialysis bags and immersed in normal autoclavedmedium spiked with metals and continuously stirred for three days. Theconcentrations of metal in the cells were measured using triplequadrupole ICP-MS (Agilent 8800, Trent Water Quality Centre). The metalsorption ratio is calculated as follows:

${Metal}\mspace{14mu}{sorption}\mspace{14mu}{ratio}{= \frac{\begin{matrix}{{{Concentration}\mspace{14mu}{of}\mspace{14mu}{metal}\mspace{14mu}{in}}\mspace{14mu}} \\{{the}\mspace{14mu}{cells}\mspace{14mu}{after}\mspace{14mu}{three}\mspace{14mu}{days}}\end{matrix}}{{Initial}\mspace{14mu}{concentration}\mspace{14mu}{of}\mspace{14mu}{metal}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{cells}}}$

D. Asymmetrical Flow Field-Flow Fractionation

Asymmetrical flow field-flow fractionation (AF4) equipped with a 300 Dapolyethersulfonate (PES) membrane, and a UV-Visible diode array detectorwas utilized (Guéguen and Cuss, 2011). The molecular weight (MW)calibration was performed with macromolecules: laser grade rhodamine B(479 Da; Acros Organics), Trypan blue (961 Da; Sigma-Aldrich), vitaminB12 (1330 Da; Sigma-Aldrich), bovine heart cytochrome C (12,400 Da;Sigma-Aldrich) and hen egg white lysozyme (14,000 Da; Fluka), wereprepared in the carrier solution. A log-log retention time versus MWcalibration curve was plotted and subsequently used to calculate the MWof samples.

E. Diffusive Gradient in Thin Films (DGTs) Technologies

Diffusive gradient in thin films (DGTs) technologies function as passivesamplers that concentrate metals from dissolved, aquatic phases (Davisonand Zhang, 1994). Currently, synthetic Chelex binding gels serve as thesite of metal interaction and concentrations; however, manufacturingChelex gels requires many chemicals and can be time consuming. To combatthis, this disclosure shows the incorporation of non-living Euglenacells and/or exudates released by Euglena as the new binding sorbent forthe passive concentration and removal of metals (FIGS. 25A and B). TheDGT samplers were composed of a binding gel layer (immobilized Euglenacells or Chelex resin), a 0.5 mm diffusive acrylamide-based gel layerand a 0.45 um cellulose nitrate filter. By embedding Euglena cells in animmobilized matrix, this disclosure shows that the need for syntheticChelex gels is omitted. The disclosure provides an all organic solutionfor metal removal. This biosorbent DGT is useful in contaminated sitesto monitor, accumulate and eventually remove metals from contaminatedaquatic sources.

The E. gracilis cells were harvested at the exponential phase andcentrifuged at 4000-5000 rpm for 6 min and washed three times withdeionized water to remove any remaining culture medium from the cells.Cells were lyophilized overnight and mechanically homogenized via mortarand pestle.

The DGT preparation was conducted in metal-free 10,000 class (ISO 7)clean room to minimize metal contamination. In order to prepare DGTresin binding gels, 3 g of Euglena cells or Chelex-100 resin was addedto 10 mL polyacrylamide gel (15% acrylamide—FisherScientific, 0.3% DGTcross-linker—DGTResearch) in a pre-cleaned polypropylene tube. 50 μl of10% (w/w) APS (>98%, FisherScientific) and 15 μM TEMED(FisherScientific) were added and mixed well. The gels were castimmediately between glass plates spaced with 0.25 mm thick, acid-bathed,polystyrene spacers. Following polymerization (60 min at 40-45° C.), thegels were hydrated in MilliQ water for at least 24 h. The MilliQ waterwas changed several times to remove any impurities and unreactedreagents. The gels were stored in 0.1M NaNO₃ solution.

DGT devices were submerged into the stirred multi-element solution (50ppb), kept at 25° C., for different periods of time (e.g. 1, 2, 3, 4,and 5 d). At each sampling interval, two DGT units were removed from thesolution. Binding gels were eluted in 1 M double distilled nitric acidfor 24 h before ICPMS analysis (Agilent 8800, Trent Water QualityCentre). Indium and rhodium were used as internal standards. Theaccuracy of the ICP-MS measurements was assessed using SLRS-5 referencewater (National Research Council, Canada). The measured metalconcentrations were within 5% of the certified values. Blankconcentrations were assessed by measuring the mass of metal present inbinding gels.

III. Results and Discussion A. Dialysis of Macromolecules

The retention characteristics of dialysis bags may vary with theoperating conditions. Thus, the rejection rate (% R) was determined byintegrity tests based on macromolecules with a known molecular weight MW(Table 21). Rejection rates averaged 99 to 100% for all macromoleculessupporting that compounds with MW greater than the smaller macromoleculeMW (i.e. Rhodamine B) are retained in the dialysis bags. As SRFA (1.1kDa) and Euglena exudates (1.6 kDa) have higher MW than Rhodamine-B,they should be preferentially retained in the dialysis bags.

TABLE 21 Retention characteristics of dialysis bags. Standard Molecularweight (kDa) % R Rhodamine B 0.5 99 Vitamin B12 1.3 99 Cytochrome C 12100 Lysozyme 14.3 100 Albumin 66 100

B. Dialysis of Euglena and SRFA

The performance of dialysis bag to retain small (SRFA), medium (Euglenaexudates) and large compounds (Euglena cells) was assessed in laboratorysettings. >99% of SRFA and Euglena exudates remained in the dialysisbags after 5 days (FIG. 26), congruent with MW results. Only 0.1 to 1.2%of the original material contained the dialysis bag was found in thedialysate after 4 days (FIG. 26). The Euglena exudates dialysate wasslightly more enriched compared to SRFA due to differences in MWdistribution (FIG. 27). The <0.5 kDa fraction was slightly more abundantin Euglena exudates than SRFA as confirmed by the higher signal reportedin the dialysate (FIG. 27).

Similarly, no Euglena cells were noticeable in the dialysate after 4days (FIG. 28C). Together these results showed that Euglena cells andexudates in dialysis bags are efficiently deployed in wastewaters ormining process water to remove contaminants.

Next, Euglena cells contained in dialysis bags were used to assess theeffects of Hg-adaptation on metal sorption. In younger generation cells(2-4 cycles), higher sorption of Pb and Cd, but not Ni and Cu, was foundin Hg-adapted cells (FIG. 29). The sorption ratio was relativelydifferent for Cd, where the Hg-adapted cells showed an approximately tentimes higher sorption ratio in comparison to normal cells. The sorptionincreased as follows:

Normal cells Ni=Cu<Cd<Pb

Hg-adapted cells Ni=Cu<Pb<Cd

By contrast, in older generation cells (11 cycles), no significantchange in metal sorption was found between normal and Hg-adapted Euglenacells (FIG. 30). For the older generation cells in both cultureconditions, the trend was as follows: Ni≈Cu<Pb<Cd.

A comparison between younger generation and older generation Hg-adaptedcells found that younger generation cells showed a 4.5-fold increase inPb sorption relative to the older generation cells (FIG. 31). Incontrast, greater metal sorption was associated with the oldergeneration for Cd in Hg-adapted cells. No significant difference in Niand Cu sorption was found between younger and older generations ofHg-adapted cells.

C. Euglena-DGT

The FTIR spectra of lyophilized E. gracilis showed several bands in the1900 to 500 cm⁻¹ range (FIG. 32), indicative of functional groups. Thebands at 1310 (COO⁻), 1394 (COO_(sym)) and 1450 cm⁻¹ (O—H bend) werewell defined in Chelex-100 compared to Euglena, confirming thatcarboxylic functional groups are dominant in Chelex-100. The absorbanceof these carboxylic bands was reduced in Euglena, indicating that theywere found in Euglena but not as abundant as in Chelex-100. The bands at1044, 1110 and 1197 cm⁻¹ (C—O_(asym)) represented carbohydrates and wasonly found in Euglena. The presence of carbohydrates was congruent withalgal metabolomics process. The amide III and Ar—OH (1232 cm⁻¹) was alsounique to Euglena. These FTIR results showed that Euglena possessed allfunctional groups (i.e. carboxylic, phenolic, amide and sulfur) requiredin metal binding.

The triplicate DGT units deployed for 72 h in 50 ug L⁻¹ showed that allmetals were accumulated onto both Euglena and Chelex binding gels (Table22). Type-B metals (Cd and Pb) were equally accumulated on both bindinggels whereas the intermediate metals (Co and Ni) were preferentiallyaccumulated on the DGT-Chelex units. Cu was more accumulated onDGT-Euglena units, without wishing to be bound by theory, could be dueto its complexation with O-, N- and S-containing functional groups. NoN- (FIG. 22) and S-containing functional groups were found on Chelexresin.

TABLE 22 Accumulated mass (M) on DGT-Euglena resin gels as a function oftime (in days). metal Al M = 73.07 t + 132 (r² = 0.95) V M = 32.86 t +11.28 (r² = 0.89) Mn M = 11.41 t + 13.91 (r² = 0.81) Co M = 10.59 t +11.54 (r² = 0.76) Ni M = 14.37 t + 17.83 (r² = 0.72) Cu M = 120.99 t +0.00 (r² = 0.93) As M = 8.10 t + 10.47 (r² = 0.71) Cd M = 20.45 t + 6.29(r² = 0.91)

Based on the diffusion properties, the mass of metal accumulated ontothe Euglena resin (M) was proportional to the exposure time(0.71<r²<0.98, FIG. 24), implying that increasing exposure timeincreased metal extraction. The diffusion coefficient values for metalsranged from 1.32×10⁻⁶ to 1.97×10⁻⁵ at 21° C. (FIG. 25). The diffusioncoefficients for Al and Cu were significantly greater in DGT-Euglenathan in DGT-Chelex. Unlike DGT-Chelex, the DGT-Euglena showed a linearaccumulation of metalloids, including As and V, showing that DGT-Euglenawas suitable for metalloid extraction.

IV. Conclusions

The Hg-adaptation experiment showed that metal sorption was dependent onthe growth conditions, cell generation, and the metal. Pb and Cdsorption, unlike Ni and Cu sorption, was enhanced in Hg-adapted cells,supporting that Hg-induced stress changed the cellular sorptionproperties. The sorption enhancement was mainly associated with cellsrecently grown in the presence of Hg, which indicated a temporal elementfor the metal sorption ability of Euglena cells following Hg-adaptation.

The application of the presented methods could ultimately be applied forthe sequestration and immobilization of metal ions in contaminatedwaters. The trapping of dissolved organic matter (DOM) within the porousdialysis bag allows for the internalization of metal ions from theenvironment and the subsequent binding to exudates and algal cellstrapped within the bag. This approach is useful for any source ofmicroorganism derived DOM larger than the molecular weight cut-off. Bytrapping cells and/or algal exudates within the dialysis bag, dualremediation effect can occur whereby there is 1) immobilization oflarger molecular weight contaminant-DOM complexes that remain in the bagand 2) the uptake/sorption of contaminant-DOM complexes by algal cells.

The immobilization of Euglena cells allows for the extraction andsequestration of metals and metalloids from the environment. Thisapproach is useful for any cellular fractions or metabolites frommicroorganisms. The skilled person would readily recognize that theDGT-Euglena is useful for any metals and metalloids that can bind to O-,N- and S-groups present on cells and metabolites.

While the present disclosure has been described with reference toexamples, it is to be understood that the scope of the claims should notbe limited by the embodiments set forth in the examples, but should begiven the broadest interpretation consistent with the description as awhole.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present disclosure is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

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1.-48. (canceled)
 49. A method of binding a target metal, the methodcomprising: contacting a solution containing a target metal with abiosorbent element; and optionally separating the complex from thesolution.
 50. The method of claim 49, wherein the biosorbent elementcomprises a substrate carrying dried Euglena biomass, or a fractionthereof, wet Euglena biomass, or a fraction thereof, or exudates ofEuglena, or a fraction thereof, in sufficient quantity to adsorb metalsfrom the solution passing therethrough.
 51. The method of claim 49,wherein the biosorbent element is a biosorbent diffusive gradient acrossa plurality of thin films.
 52. The method of claim 49, wherein thebiosorbent element binds metals comprising one or more of silver, gold,aluminum, arsenic, barium, beryllium, bismuth, calcium, cadmium, cobalt,chromium, copper, iron, potassium, lithium, magnesium, manganese,molybdenum, sodium, nickel, phosphorus, platinum, palladium, lead,antimony, selenium, tin, strontium, thallium, titanium, uranium,vanadium, tungsten, yttrium, zinc, scandium, lanthanum, rare earthelements, and divalent transition metals.
 53. The method of claim 49,wherein the biosorbent element is for use in remediation of wastewater.54. The method of claim 53, wherein the biosorbent element is contactedwith the wastewater before the wastewater contacts activated carbon. 55.The method of claim 53, wherein the wastewater is domestic wastewater,urban wastewater, industrial wastewater or combinations thereof.
 56. Themethod of claim 55, wherein the industrial wastewater comprises effluentfrom a mining operation.
 57. The method of claim 50, wherein the driedEuglena biomass, or a fraction thereof, or the wet Euglena biomass, or afraction thereof, or the exudates of a culture of Euglena, or a fractionthereof, comprises glutathione, metallothioneins, phytochelatins,polyphosphates, polysaccharides, or combinations thereof.
 58. The methodof claim 50, wherein the dried Euglena biomass, or a fraction thereof,or the wet Euglena biomass, or a fraction thereof, or the exudates of aculture of Euglena, or a fraction thereof, is contained in a dialysiscontainer or dialysis bag.
 59. The method of claim 50, wherein the driedEuglena biomass, or a fraction thereof, or the wet Euglena biomass, or afraction thereof, or the exudates of a culture of Euglena, or a fractionthereof, is embedded in a diffusive gradient in a plurality of thinfilms, optionally a diffusion gradient technology (DGT).
 60. The methodof claim 50, wherein the dried Euglena biomass, or a fraction thereof,or the wet Euglena biomass, or a fraction thereof, or the exudates of aculture of Euglena, or a fraction thereof, is spherical and/orgelatinous.
 61. The method of claim 60, wherein the biosorbent elementfurther comprises an immobilizing matrix to encapsulate the driedEuglena biomass, or a fraction thereof, or the wet Euglena biomass, or afraction thereof, or the exudates of a culture of Euglena, or a fractionthereof.
 62. The method of claim 61, wherein the immobilizing matrixcomprises a resin or a polymer plastic.
 63. The method of claim 61,wherein the immobilizing matrix comprises agar, agarose, alginate,carrageenan, cellulose, chitosan, polystyrene, polyurethane, polyvinyl,or combinations thereof.
 64. The method of claim 63, wherein theimmobilizing matrix comprises sodium alginate.